WO2012050125A1 - モナティンの製造方法 - Google Patents
モナティンの製造方法 Download PDFInfo
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- WO2012050125A1 WO2012050125A1 PCT/JP2011/073416 JP2011073416W WO2012050125A1 WO 2012050125 A1 WO2012050125 A1 WO 2012050125A1 JP 2011073416 W JP2011073416 W JP 2011073416W WO 2012050125 A1 WO2012050125 A1 WO 2012050125A1
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- amino acid
- monatin
- aminotransferase
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/12—Radicals substituted by oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
- C12Y206/01001—Aspartate transaminase (2.6.1.1), i.e. aspartate-aminotransferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01003—Oxaloacetate decarboxylase (4.1.1.3)
Definitions
- the present invention relates to a method for producing monatin using L-amino acid aminotransferase.
- monatin is produced by synthesizing indole-3-pyruvic acid (hereinafter referred to as “IPA” if necessary) from L-tryptophan (L-Trp), and obtaining the resulting IPA and pyrubin.
- 4R isomer of 4- (indol-3-yl-methyl) -4-hydroxy-2-oxoglutaric acid (hereinafter referred to as “4R-IHOG” if necessary) was synthesized from the acid, and then the 4R obtained
- the following method (conventional method (1)) for producing 2R, 4R-monatin by subjecting -IHOG to oximation reaction, reduction reaction and epicrystallization crystallization method is known (see Patent Document 2) ).
- the aldolase step (second step) is an equilibrium reaction, it cannot always be said that a satisfactory yield is obtained.
- Patent Document 3 a method for producing 2R, 4R-monatin by a one-pot enzymatic reaction
- An object of the present invention is to provide a method for producing monatin with good yield.
- L-amino acid aminotransferase As a result of intensive studies, the present inventors have found that the above problems can be solved by using L-amino acid aminotransferase, and have completed the present invention.
- An L-amino acid aminotransferase that acts on 4R-IHOG has not been known so far.
- the present invention is as follows.
- a process for producing 2S, 4R-monatin or a salt thereof comprising contacting 4R-IHOG with an L-amino acid aminotransferase in the presence of an L-amino acid to produce 2S, 4R-monatin.
- the production method of [1] further comprising decomposing a keto acid produced from the L-amino acid by the action of the L-amino acid aminotransferase by contacting with decarboxylase.
- the production method of [1] wherein the L-amino acid is L-aspartic acid.
- L-amino acid aminotransferase is selected from Arthrobacter, Bacillus, Candida, Corynebacterium, Roderomyces, Micrococcus, Microbacterium, Nocardia, Pseudomonas, Rhizobium, Stenotropho Monas spp., Diedia sp., Ocrobactrum sp., Breven Dimonas sp., Burkholderia sp., Carnimonas sp., Yarrowia sp., Clostridium sp., Deinococcus sp., Eubacterium sp., Lactobacillus sp., Methanothermobacter sp.
- [1] which is derived from a microorganism belonging to the genus Rhodococcus, Saccharomyces, Saccharophagas, Synorizobium, Thermoanaerobacter, Thermotoga, or Thermus.
- L-amino acid aminotransferase is selected from the group consisting of Arthrobacter sp., Bacillus artitudinis, Bacillus cellulolyticus, Bacillus pumilus, Bacillus sp. ⁇ Ammonia Genes, Corynebacterium glutamicum, Roderomyces elongisporus, Micrococcus luteus, Microbacterium sp. ⁇ Synxantha, Pseudomonas taetororens, Pseudomonas sp, Rhizobium radiobacter, Rhizobium et P., Stenotrophomonas sp., Diezia maris, Ocrobactrum pseudogrignonens, Breven dimonas diminuta, Burkholderia sp, Carnimonas sp., Yarrowia lipolytica, Clostridium cellulolyticum, Deinococcus geothermaris, Eubacterium Recir
- L-amino acid aminotransferase is SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, Sequence number 83, sequence number 85, sequence number 87, sequence number 89, sequence number 91, sequence number 93, sequence number 95, sequence number 97, sequence number 99, sequence number 101, sequence number 103, sequence number 105, sequence number 107.
- L-amino acid aminotransferase is 39, 109, 128, 150, 258, 287, 288, 289, 303, 358 in the amino acid sequence represented by SEQ ID NO: 2,
- a production method of [7] comprising a mutation of one or more amino acid residues selected from the amino acid residues at position 431.
- [17] A process for producing 2R, 4R-monatin or a salt thereof comprising the following (I) and (II): (I) producing 2S, 4R-monatin by the method of [1]; and (II) isomerizing 2S, 4R-monatin to produce 2R, 4R-monatin. [18] The production method of [17], wherein 2S, 4R-monatin is isomerized in the presence of an aromatic aldehyde. [19] The production method of [17], wherein the salt is a sodium salt or a potassium salt.
- L-amino acid aminotransferase which is a protein selected from the group consisting of the following (A) to (D): (A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 53, or SEQ ID NO: 61; (B) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 53, or SEQ ID NO: 61; (C) It consists of an amino acid sequence showing 90% or more identity to the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 53, or SEQ ID NO: 61, and has L-amino acid aminotransferase activity And (D) selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues in the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 53, or SEQ ID NO: 61.
- a protein comprising an amino acid sequence containing a mutation of one or several amino acid residues and having L-amino acid aminotransferase activity.
- L-amino acid aminotransferase is 39, 109, 128, 150, 258, 287, 288, 289, 303, 358 in the amino acid sequence represented by SEQ ID NO: 2,
- L-amino acid aminotransferase of [20] comprising a mutation of one or more amino acid residues selected from the amino acid residues at position 431.
- the method of the present invention can contribute to the improvement of the yield of 2R, 4R-monatin by producing 2S, 4R-monatin from 4R-IHOG with a good yield using L-amino acid aminotransferase.
- the method of the present invention also eliminates the need to use expensive D-amino acids (such as D-Asp) as substrates when producing 2S, 4R-monatin from IHOG, or produces D-amino acids from L-amino acids. There is an advantage that it is not necessary to add an enzyme such as racemase.
- the method of the present invention further includes not only a reaction that generates 2S, 4R-monatin from 4R-IHOG (third step), but also a reaction that generates IPA from L-Trp (first step), and 4R-IHOG from IPA.
- the overall reaction equilibrium can be defined in the third step, and the reaction equilibrium in the second step can be greatly shifted in the direction of 4R-IHOG production. it can.
- the method of the present invention makes it possible to produce 2S, 4R-monatin in a very good yield by avoiding by-production of L-Trp (progress of reverse reaction in the first step). To do.
- FIG. 1 is a diagram showing an example of the production method of the present invention. Trp: tryptophan; IPA: indole-3-pyruvic acid; IHOG: 4- (indol-3-yl-methyl) -4-hydroxy-2-oxoglutaric acid; monatin: 4- (indol-3-yl-methyl)- 4-hydroxy-glutamic acid.
- FIG. 2 is a diagram showing an example of the production method of the present invention. The abbreviations are the same as in FIG. FIG. 3 shows a preferred example of the production method of the present invention.
- FIG. 4 is a diagram showing a 2S, 4R-monatin production reaction from L-Trp on a 400 ml scale using an L-amino acid aminotransferase mutant (ID166).
- ID166 L-amino acid aminotransferase mutant
- FIG. 5 is a diagram showing a 2S, 4R-monatin production reaction from L-Trp on an 80 ml scale using an L-amino acid aminotransferase mutant (ID189). Abbreviations are the same as in FIG.
- FIG. 5 is a diagram showing a 2S, 4R-monatin production reaction from L-Trp on an 80 ml scale using an L-amino acid aminotransferase mutant (ID296). Abbreviations are the same as in FIG.
- the present invention provides a method (1) for producing 2S, 4R-monatin or a salt thereof.
- the production method of the present invention includes (1-1) a method for producing 2S, 4R-monatin from 4R-IHOG, (1-2) a method for producing 2S, 4R-monatin from IPA and pyruvic acid, (1-3 ) A method for producing 2S, 4R-monatin from tryptophan.
- 4R-IHOG is contacted with an L-amino acid aminotransferase in the presence of an L-amino acid to produce 2S, 4R-monatin.
- an L-amino acid aminotransferase is contacted with an L-amino acid aminotransferase in the presence of an L-amino acid to produce 2S, 4R-monatin.
- L-amino acid is not particularly limited as long as it is an L-amino acid whose amino group can be transferred to the target substrate 4R-IHOG by L-amino acid aminotransferase.
- L-amino acids various L-amino acids such as L- ⁇ -amino acids are known.
- L-amino acids include L-aspartic acid, L-alanine, L-lysine, L-arginine, L-histidine, L-glutamic acid, L-asparagine, L-glutamine, L-serine.
- the L-amino acid may be added to the reaction solution in the form of a salt.
- concentration of the L-amino acid in the reaction solution is, for example, 1 mM to 3M, preferably 20 mM to 1M, and more preferably 100 mM to 500 mM.
- the L-amino acid aminotransferase may be a protein derived from a microorganism such as a bacterium, actinomycetes, or yeast. Microorganisms are classified according to methods well known in the art, for example, NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwwtc.g? 91347).
- NCBI National Center for Biotechnology Information
- microorganisms from which L-amino acid aminotransferases are derived include the genera Arthrobacter, Bacillus, Candida, Corynebacterium, Rodderomyces, Micrococcus (Micrococcus), Microbacterium, Nocardia, Pseudomonas, Rhizobium, Stenotrohomonas, Dietiac Dietia Genus, Brevenundimonas (Brevun genus Imonas, Burkholderia, Carnimonas, Yarrowia, Clostridium, Deinococcus, Eubacterium lacteus, Eubacterium lactus , Methanococcus genus, Methanotherbacter genus, Hormidium genus, Pyrococcus genus, Rhodococcus genus, Saccharomyces saccharomyces Rhizobium (Sinorhizobium) genus Thermoanaerobacter Citrobacter (Thermoana
- examples of microorganisms belonging to the genus Arthrobacter include Arthrobacter sp.
- microorganisms belonging to the genus Bacillus include Bacillus altitudinis, Bacillus cellulolyticus, Bacillus pumilus, Bacillus sp. (Bacillus sp.). .
- Examples of microorganisms belonging to the genus Candida include Candida norvegensis and Candida inconspicua.
- microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes and Corynebacterium glutamicum.
- microorganisms belonging to the genus Roderomyces include Roderomyces elongis porus.
- microorganisms belonging to the genus Micrococcus include Micrococcus luteus.
- An example of a microorganism belonging to the genus Microbacterium is Microbacterium sp.
- Examples of microorganisms belonging to the genus Nocardia include Nocardia globulara.
- Pseudomonas chlororaphis for example, Pseudomonas chlororaphis subsp. Pseudomonas fragii, Pseudomonas putida, Pseudomonas synxanta, Pseudomonas taetrorens, Pseudomonas tatrolens Pseudomonas sp. (Pseudomonas sp.), And the like.
- microorganisms belonging to the genus Rhizobium include Rhizobium radiobacter and Rhizobium sp.
- microorganisms belonging to the genus Stenotrohomomonas include Stenotrophomonas sp.
- An example of a microorganism belonging to the genus Diedia is Dietia maris.
- Examples of microorganisms belonging to the genus Okrobactrum include Okrobactrum pseudogrignonense.
- microorganisms belonging to the genus Brevendimonas include Brevundimonas diminuta.
- microorganisms belonging to the genus Burkholderia include Burkholderia sp.
- An example of a microorganism belonging to the genus Carnimonas is Carnimonas sp.
- Examples of microorganisms belonging to the genus Yarrowia include Yarrowia lipolytica.
- microorganisms belonging to the genus Clostridium include Clostridium cellulolyticum.
- Examples of microorganisms belonging to the genus Deinococcus include Deinococcus geothermalis.
- microorganisms belonging to the genus Eubacterium include Eubacterium rectal.
- Examples of microorganisms belonging to the genus Lactobacillus acidophilus include Lactobacillus acidophilus.
- microorganisms belonging to the genus Methanococcus include Methanococcus jannaschii.
- Examples of microorganisms belonging to the genus Methanothermobacter include Methanothermobacter thermotrophicus.
- Examples of microorganisms belonging to the genus Hormidium include Hormidium lapideum.
- Examples of microorganisms belonging to the genus Pyrococcus include Pyrococcus horikoshii.
- Examples of microorganisms belonging to the genus Rhodococcus include Rhodococcus erythropolis.
- Examples of microorganisms belonging to the genus Saccharomyces include Saccharomyces cerevisiae.
- Examples of microorganisms belonging to the genus Saccharophagas include Saccharophagas degradans.
- Examples of microorganisms belonging to the genus Sinorhizobium include Sinorhizobium merilloti.
- microorganisms belonging to the genus Thermoanaerobacter include Thermoanaerobacter tengcongenesis.
- microorganisms belonging to the genus Thermotoga include Thermotoga maritima.
- An example of a microorganism belonging to the genus Thermus is Thermus thermophilus.
- the L-amino acid aminotransferase can be a natural protein or an artificial mutant protein.
- Such L-amino acid aminotransferases include SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77.
- L-amino acid aminotransferase activity refers to the activity of transferring the amino group of an L-amino acid to the target substrate 4R-IHOG to produce 2S, 4R-monatin, the target compound having an amino group.
- the L-amino acid aminotransferase is 80% or more, preferably 90% or more, more preferably 95%, particularly preferably 98% or more or 99% with respect to the amino acid sequence represented by SEQ ID NO: 2.
- the homology between the amino acid sequence and the base sequence can be determined by, for example, the algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Can be determined. Since programs called BLASTP and BLASTN have been developed based on this algorithm BLAST (see http://www.ncbi.nlm.nih.gov), these programs are used with default settings, and amino acid sequences and You may calculate the homology of a base sequence.
- the L-amino acid aminotransferase comprises SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 75, Sequence number 77, sequence number 83, sequence number 85, sequence number 87, sequence number 89, sequence number 91, sequence number 93, sequence number 95, sequence number 97, sequence number 99, sequence number 101, sequence number 103, sequence number 105, the amino acid sequence represented by SEQ ID NO: 107, SEQ ID NO: 109, or SEQ ID NO: 111 comprises an amino acid sequence containing a mutation (eg, deletion, substitution, addition and insertion) of one or several amino acid residues And a protein having L-amino acid aminotransferase activity.
- SEQ ID NO: 111 comprises an amino acid sequence containing a mutation (eg, deletion, substitution, addition and insertion)
- the mutation of one or several amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions.
- the term “one or several” indicates a range that does not significantly impair the three-dimensional structure and activity of the protein.
- the number indicated by the term “one or several” in the case of protein is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5.
- Such a mutation may be caused by a naturally occurring mutation (mutant or variant) based on individual differences, species differences, and the like of microorganisms holding a gene encoding L-amino acid aminotransferase.
- the position of the amino acid residue to be mutated in the amino acid sequence is obvious to those skilled in the art. Specifically, those skilled in the art will compare 1) the amino acid sequences of a plurality of proteins having the same activity (eg, the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequences of other L-amino acid aminotransferases). 2) reveal the relatively conserved regions and the relatively unconserved regions, then 3) from the relatively conserved regions and the relatively unconserved regions, respectively, Since regions that can play an important role in function and regions that cannot play an important role in function can be predicted, the correlation between structure and function can be recognized. Accordingly, those skilled in the art can specify the position of the amino acid residue to be mutated in the amino acid sequence of L-amino acid aminotransferase.
- amino acid residue substitution may be a conservative substitution.
- conservative substitution refers to the replacement of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art.
- such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g.
- the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine. Or a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.
- the L-amino acid aminotransferase is SEQ ID NO: 1, SEQ ID NO: 47, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 74, Sequence number 76, sequence number 82, sequence number 84, sequence number 86, sequence number 88, sequence number 90, sequence number 92, sequence number 94, sequence number 96, sequence number 98, sequence number 100, sequence number 102, sequence number 104, encoded by DNA that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 106, SEQ ID NO: 108, or SEQ ID NO: 110, and has L-amino acid aminotransferase activity It may be a protein.
- “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to clearly quantify such conditions, for example, polynucleotides having high homology (eg, identity), for example, 80%, preferably 90% or more, more preferably 95 %, Particularly preferably 98% or more of the polynucleotides having a homology hybridize, and polynucleotides having a lower homology do not hybridize. Specifically, such conditions include hybridization at about 45 ° C. in 6 ⁇ SSC (sodium chloride / sodium citrate), followed by 50 ⁇ 0.2 ⁇ SSC in 0.1% SDS. One or more washings at ⁇ 65 ° C. may be mentioned.
- SSC sodium chloride / sodium citrate
- L-amino acid aminotransferase is in the amino acid sequence represented by SEQ ID NO: 2, 39, 109, 128, 150, 258, 287, 288, 289, 303, 358 It may be an L-amino acid aminotransferase mutant in which one or more (eg, 1 or 2) arbitrary amino acid residues selected from the amino acid residues at position 431 and 431 are mutated by substitution or the like .
- L-amino acid aminotransferase variants are those comprising one or more (eg, 1 or 2) substitutions selected from the group consisting of: 1) substitution of lysine at position 39 with arginine; 2) substitution of serine at position 258 with glycine; 3) replacement of glutamine at position 287 with glutamic acid; 4) replacement of threonine at position 288 with glycine; 5) Substitution of isoleucine at position 289 with alanine; 6) substitution of aspartic acid at position 109 with glycine; 7) replacement of histidine at position 150 with tyrosine; 8) substitution of phenylalanine at position 303 with leucine; 9) substitution of aspartic acid at position 358 with tyrosine; 10) substitution of serine at position 431 with threonine; and 11) substitution of glutamic acid at position 128 with glycine.
- substitutions selected from the group consisting of: 1) substitution of lysine at position
- substitution of one or more (eg, 1 or 2) arbitrary amino acid residues for example, the following combinations of mutations can be introduced, but amino acids that can be used in the present invention The combination of substitutions is not limited to the following.
- the contact of 4R-IHOG with L-amino acid aminotransferase is carried out by using 4R-IHOG and L-amino acid aminotransferase (extracted enzyme) extracted from the L-amino acid aminotransferase-producing bacterium in the reaction solution.
- L-amino acid aminotransferase-producing bacteria include bacteria that naturally produce L-amino acid aminotransferase (eg, the microorganism described above), and transformants that express L-amino acid aminotransferase.
- the extracted enzyme for example, a purified enzyme, a crude enzyme, an immobilized enzyme, a culture broth, a culture broth-treated product (eg, a fraction containing L-amino acid aminotransferase prepared from the producing bacteria, the production described above) Crushed and lysate of fungi).
- a culture broth-treated product eg, a fraction containing L-amino acid aminotransferase prepared from the producing bacteria, the production described above
- the treatment for obtaining a culture broth-treated product from the culture broth include heat treatment (42 to 80 ° C., pH 3 to 12, 1 minute to 24 hours), solvent treatment (eg, xylene, toluene, ethanol, isopropyl alcohol).
- Surfactants eg, Tween 20, Triton X-100
- lytic enzyme treatment eg, lysozyme treatment.
- the temperature in this case is 4 ° C. to 60 ° C., preferably 20 ° C. to 37 ° C.
- the pH can be set to 3-12, preferably 7-9.
- the time can be set to about 5 minutes to 20 days, preferably about 1 hour to 7 days. Aeration stirring may or may not be performed while holding the broth.
- contact of 4R-IHOG with L-amino acid aminotransferase is achieved by allowing 4R-IHOG and L-amino acid aminotransferase-producing bacteria to coexist in a reaction solution (eg, culture solution). it can.
- a reaction solution eg, culture solution
- the reaction solution used in the production method (1) of the present invention is not particularly limited as long as the target reaction proceeds.
- water or a buffer solution is used.
- the buffer solution include Tris buffer solution, phosphate buffer solution (eg, KH 2 PO 4 ), carbonate buffer solution, borate buffer solution, and acetate buffer solution.
- the concentration of the buffer may be, for example, 0.1 mM to 10M, preferably 1 mM to 1M.
- a culture solution may be used as a reaction solution.
- Such a culture solution can be prepared using, for example, a medium described later.
- the reaction solution used in the production method of the present invention may further contain pyridoxal phosphate (PLP) as a coenzyme.
- PLP may be added to the reaction solution in the form of a salt.
- the concentration of PLP in the reaction solution is, for example, 1 ⁇ M to 100 mM, and preferably 10 ⁇ M to 1 mM.
- an effect of generating 2R, 4R-monatin from 2S, 4R-monatin can also be expected by an isomerization reaction that can be catalyzed by PLP (see, for example, Example 11).
- the pH of the reaction solution used in the production method (1) of the present invention is not particularly limited as long as the target reaction proceeds.
- the pH is 5 to 10, preferably 6 to 9, and more preferably 7 to 8.
- the reaction temperature in the production method (1) of the present invention is not particularly limited as long as the target reaction proceeds, and is, for example, 10 to 50 ° C., preferably 20 to 40 ° C., more preferably 25 to 35 ° C.
- the reaction time in the production method (1) of the present invention is not particularly limited as long as it is sufficient to produce 2S, 4R-monatin.
- it is 2 to 100 hours, preferably 4 to 50 hours, more preferably Is 8-25 hours.
- this transformant When a transformant that expresses L-amino acid aminotransferase is used as the L-amino acid aminotransferase-producing bacterium, this transformant, for example, produces an expression vector for L-amino acid aminotransferase, and then expresses this expression vector. It can be produced by introducing a vector into a host.
- a transformant expressing L-amino acid aminotransferase can be obtained by preparing an expression vector incorporating a DNA having the nucleotide sequence represented by SEQ ID NO: 1 and introducing it into an appropriate host.
- Examples of hosts for expressing L-amino acid aminotransferase include Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (eg, Corynebacterium glutamicum), and Bacillus genus.
- Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (eg, Corynebacterium glutamicum), and Bacillus genus.
- Various prokaryotic cells including bacteria (eg, Bacillus subtilis), bacteria belonging to the genus Saccharomyces (eg, Saccharomyces cerevisiae), bacteria belonging to the genus Pichia (eg, Pichia stipitis) , Aspergillus oryzae (eg, Aspergillus oryzae ) It is possible to use various eukaryotic cells including.
- a strain lacking a predetermined gene may be used.
- genes that can be deleted include AspC, host-derived L-amino acid aminotransferase, host-derived aldolase, and host-derived deaminase.
- the transformant include a transformant having a vector in the cytoplasm and a transformant having a target gene introduced on the genome.
- the L-amino acid aminotransferase-producing bacterium can be cultured in a medium having the composition described below using a predetermined culture apparatus (eg, test tube, flask, jar fermenter, etc.).
- the culture conditions can be set as appropriate.
- the culture temperature may be 25 ° C. to 37 ° C.
- the pH may be 6.5 to 7.5
- the culture time may be 1 h to 100 h.
- the dissolved oxygen concentration (DO value) in the culture solution may be used as a control index.
- the culture may be batch culture or fed-batch culture.
- a solution serving as a sugar source or a solution containing phosphoric acid can be added continuously or discontinuously to the culture solution to continue the culture.
- the host to be transformed is as described above, but in detail about E. coli, it can be selected from Escherichia coli JM109 strain, DH5 ⁇ strain, HB101 strain, BL21 (DE3) strain, etc.
- a method for performing transformation and a method for selecting transformants are also described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001/01/15) and the like.
- a method for producing transformed E. coli and producing a predetermined enzyme using the same will be described more specifically as an example.
- Promoters that express DNA encoding L-amino acid aminotransferase are usually E. coli. Promoters used for heterologous protein production in E. coli can be used, such as PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, T5 promoter, etc. A strong promoter is mentioned, and PhoA, PhoC, and lac are preferable.
- the vector examples include pUC (eg, pUC19, pUC18), pSTV, pBR (eg, pBR322), pHSG (eg, pHSG299, pHSG298, pHSG399, pHSG398), RSF (eg, RSF1010), pACYC (eg, pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), and derivatives thereof may be used.
- phage DNA vectors may be used.
- an expression vector containing a promoter and capable of expressing the inserted DNA sequence may be used.
- the vector may be pUC, pSTV, pMW.
- a terminator that is a transcription termination sequence may be linked downstream of the L-amino acid aminotransferase gene.
- examples of such terminators include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and E. coli trpA gene terminator.
- a so-called multicopy type is preferable, and a plasmid having a replication origin derived from ColE1, such as a pUC series plasmid, a pBR322 series plasmid or a derivative thereof, is used.
- a plasmid having a replication origin derived from ColE1 such as a pUC series plasmid, a pBR322 series plasmid or a derivative thereof.
- the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion, addition and / or inversion.
- the “modification” here includes modification by mutation treatment, UV irradiation, natural mutation, or the like.
- the vector has a marker such as an ampicillin resistance gene in order to select transformants.
- a marker such as an ampicillin resistance gene
- an expression vector having a strong promoter is commercially available (eg, pUC system (manufactured by Takara Bio Inc.), pPROK system (manufactured by Clontech), pKK233-2 (manufactured by Clontech)).
- the medium a medium usually used for culturing Escherichia coli such as M9-casamino acid medium and LB medium may be used.
- the culture medium may contain a predetermined carbon source, nitrogen source, and coenzyme (eg, pyridoxine hydrochloride).
- coenzyme eg, pyridoxine hydrochloride
- the culture conditions and production induction conditions are appropriately selected according to the type of the marker, promoter, host fungus and the like used.
- L-amino acid aminotransferase recovers L-amino acid aminotransferase-producing bacteria and then crushes (eg, sonication, homogenization) or dissolves (eg, lysozyme treatment) to give crushed materials and lysates. Can be obtained as By subjecting such crushed material and lysate to extraction, precipitation, filtration, column chromatography, and the like, a purified enzyme, crude enzyme, or L-amino acid aminotransferase-containing fraction can be obtained.
- the production method of the present invention comprises contacting keto acid (R-COCOOH) generated from L-amino acid (eg, L- ⁇ -amino acid) by the action of L-amino acid aminotransferase with decarboxylase. Further comprising decomposing (see reaction 1 ').
- keto acid R-COCOOH
- L-amino acid eg, L- ⁇ -amino acid
- decarboxylase e.g., L- ⁇ -amino acid
- decarboxylase e.g., L-amino acid aminotransferase with decarboxylase
- decomposing see reaction 1 '.
- the decarboxylase used in the present invention is an enzyme that catalyzes the decarboxylation reaction of keto acid.
- Decarboxylation by decarboxylase can be irreversible.
- Various enzymes are known as decarboxylase used for irreversible decarboxylation of keto acid. For example, Pseudomonas stutzeri oxaloacetate decarboxylase (Arch Biochem Biophys., 365) 17-24, 1999), and Zymomonas mobilis-derived pyruvate decarboxylase (Applied Microbiology and Biotechnology, 17, 152-157, 1983).
- the production method of the present invention comprises contacting oxaloacetate (OAA) produced from L-aspartic acid (L-Asp) by the action of L-amino acid aminotransferase with oxaloacetate decarboxylase to form pyrubin.
- Generating acid (PA) (see reaction 1 ′′).
- PA oxaloacetate
- L-Asp L-aspartic acid
- PA oxaloacetate
- L-aspartic acid may be added to the reaction solution in the form of a salt.
- the concentration of L-aspartic acid in the reaction solution is 1 mM to 3 M, preferably 20 mM to 1 M, more preferably 100 mM to 500 mM.
- the oxaloacetate decarboxylase used in the present invention is an enzyme that produces pyruvic acid by catalyzing the decarboxylation reaction of oxaloacetate.
- the decarboxylation reaction with oxaloacetate decarboxylase can be irreversible.
- Various enzymes are known as oxaloacetate decarboxylase used for irreversible decarboxylation of oxaloacetate.
- oxaloacetate decarboxylase examples include, for example, Pseudomonas stutzeri oxaloacetate decarboxylase (Arch Biochem Biophys., 365, 17-24, 1999), Klebsiella aerogenes (Klebsiella aerogenes) Examples include acetate decarboxylase (FEBS Lett., 141, 59-62, 1982), and oxaloacetate decarboxylase (Biochim Biophys Acta., 957, 301-311, 1988) derived from Sulfolobus solfatricus.
- decarboxylase When decarboxylase is used in the production of 2S, 4R-monatin from 4R-IHOG, the contact of the keto acid produced from the L-amino acid with the decarboxylase was extracted from the keto acid and the decarboxylase producer This can be achieved by allowing a decarboxylase (extracting enzyme) or a decarboxylase producing bacterium to coexist in a reaction solution (eg, culture solution).
- a decarboxylase extracting enzyme
- a decarboxylase producing bacterium examples include bacteria that naturally produce decarboxylase and transformants that express decarboxylase.
- Examples of the extracted enzyme include purified enzyme, crude enzyme, immobilized enzyme, culture broth, and culture broth-treated product (eg, decarboxylase-containing fraction prepared from the above-mentioned producing bacteria, crushed material and lysate of the above-mentioned producing bacteria).
- Examples of the treatment for obtaining a culture broth-treated product from the culture broth include heat treatment (42 to 80 ° C., pH 3 to 12, 1 minute to 24 hours), solvent treatment (eg, xylene, toluene, ethanol, isopropyl alcohol). , Surfactants (eg, Tween 20, Triton X-100), and lytic enzyme treatment (eg, lysozyme treatment).
- the temperature in this case is 4 ° C. to 60 ° C., preferably 20 ° C. to 37 ° C.
- the pH can be set to 3-12, preferably 7-9.
- the time can be set to about 5 minutes to 20 days, preferably about 1 hour to 7 days. Aeration stirring may or may not be performed while holding the broth.
- L-amino acid aminotransferase and decarboxylase are used in the production of 2S, 4R-monatin from 4R-IHOG, L-amino acid aminotransferase and decarboxylase are provided in the reaction in the following manner: Also good.
- the L-amino acid aminotransferase and decarboxylase producing bacterium may be a transformant.
- a transformant i) an L-amino acid aminotransferase expression vector is introduced into a decarboxylase producing bacterium, and ii) a decarboxylase expression vector is introduced into the L-amino acid aminotransferase producing bacterium.
- L-amino acid aminotransferase and decarboxylase expression vectors include i ′) a first polynucleotide encoding L-amino acid aminotransferase, and a first polynucleotide operably linked to the first polynucleotide.
- the Expression vectors containing promoter (vector capable of expressing the polycistronic mRNA) are exemplified.
- the first polynucleotide encoding L-amino acid aminotransferase may be located upstream or downstream of the second polynucleotide encoding decarboxylase.
- IPA and pyruvic acid are condensed to produce 4R-IHOG. It may further include.
- the condensation of IPA and pyruvic acid can be performed by an organic chemical method and an enzymatic method using an aldolase. Methods for producing 4R-IHOG by condensing IPA and pyruvic acid by an organic chemical method are disclosed in, for example, International Publication No. 2003/059865 and US Patent Application Publication No. 2008/0207920.
- Methods for producing 4R-IHOG by condensing IPA and pyruvic acid by an enzymatic method using aldolase include, for example, International Publication No. 2003/056026, Japanese Patent Application Laid-Open No. 2006-204285, and US Patent Application Publication No. 2005/0244939. And International Publication No. 2007/103989.
- these methods can be used to prepare 4R-IHOG from IPA and pyruvate.
- IPA used for the preparation of 4R-IHOG is an unstable compound. Therefore, the condensation of IPA and pyruvic acid may be performed in the presence of an IPA stabilizing factor.
- the IPA stabilizing factor include superoxide dismutase (eg, see International Publication No. 2009/0283338) and mercaptoethanol (see, eg, International Publication No. 2009/0283338).
- superoxide dismutase eg, see International Publication No. 2009/0283338
- mercaptoethanol see, eg, International Publication No. 2009/0283338
- transformant that expresses superoxide dismutase is disclosed in International Publication No. 2009/0283338, such a transformant may be used in the method of the present invention.
- the reaction for producing 4R-IHOG from IPA and pyruvic acid and the reaction for producing 2S, 4R-monatin from 4R-IHOG may proceed separately or in parallel. These reactions may be carried out in one reaction tank. When performing these reactions in one reaction vessel, these reactions can be performed by adding the substrate and enzyme sequentially or simultaneously.
- a reaction for producing 4R-IHOG from IPA and pyruvate by an enzymatic method using aldolase and a reaction for producing 2S, 4R-monatin from 4R-IHOG by L-amino acid aminotransferase
- 1) IPA, pyruvate and aldolase, and (2) L-amino acid and L-amino acid aminotransferase may be added sequentially or simultaneously in one reaction vessel.
- Pyruvate may be added to the reaction solution in the form of a salt (eg, sodium salt). Pyruvate may be added to the reaction solution by any method (eg, batch method, feed method).
- the concentration of pyruvic acid in the reaction solution may be, for example, 0.1 mM to 10M, preferably 1 mM to 1M.
- the production method of the present invention is used in combination with the above reaction 1 ′′ as follows.
- pyruvic acid generated irreversibly from oxaloacetic acid is utilized for the preparation of 4R-IHOG.
- at least a part of pyruvic acid used for the production of 4R-IHOG can be derived from pyruvic acid produced from oxaloacetate by the action of oxaloacetate decarboxylase.
- aldolase When aldolase is used in the production of 4R-IHOG from IPA and pyruvate, contact with IPA and pyruvate with aldolase is an aldolase (extract enzyme) extracted from IPA and pyruvate, and aldolase producing bacteria. Alternatively, it can be achieved by allowing an aldolase-producing bacterium to coexist in a reaction solution (eg, culture solution). Examples of aldolase-producing bacteria include bacteria that naturally produce aldolase, and transformants that express aldolase.
- Examples of the extracted enzyme include purified enzyme, crude enzyme, immobilized enzyme, culture broth, and culture broth-treated product (eg, aldolase-containing fraction prepared from the above producing bacteria, crushed material and lysate of the above producing bacteria).
- Examples of the treatment for obtaining the culture broth treated product from the culture broth include heat treatment (42 ° C. to 80 ° C., pH 3 to 12, 1 minute to 24 hours), solvent treatment (eg, xylene, toluene), surfactant treatment. Is mentioned.
- the culture broth may be used under conditions of 4 ° C. to 60 ° C., pH 3 to 12, 5 minutes to 20 days (with / without aeration stirring).
- the aldolase-producing bacterium may further express other enzymes (eg, superoxide dismutase, L-amino acid aminotransferase, decarboxylase).
- other enzyme-producing bacteria may coexist in the reaction solution.
- the reaction solution for example, those described in the production method (1-1) of the present invention can be used.
- the aldolase, L-amino acid aminotransferase and decarboxylase producing bacterium may be a transformant. Expression of aldolase, L-amino acid aminotransferase and decarboxylase may be performed using the same transformant, or may be performed by combining two transformants, or three different transformations. It may be expressed in the body. When the aldolase, L-amino acid aminotransferase and decarboxylase genes are expressed in the same transformant, these genes may be incorporated into the chromosome, or the aldolase, L-amino acid aminotransferase and decarboxylase genes may be incorporated into one vector. Genes may be linked.
- an L-amino acid aminotransferase expression vector may be introduced into a decarboxylase and aldolase producing bacterium, or an L-amino acid aminotransferase first expression vector and a decarboxylase and aldolase second expression vector. Alternatively, it may be introduced into the host microorganism.
- expression vectors for aldolase, L-amino acid aminotransferase and decarboxylase include i ′) a first polynucleotide encoding L-amino acid aminotransferase, and a first operably linked to the first polynucleotide.
- An expression vector comprising a third expression unit composed of an expression unit, a third polynucleotide encoding an aldolase, and a third promoter operably linked to the third polynucleotide, and ii ′) L-amino acid aminotransferase
- reaction temperature a condition in which reaction temperature, pH, and time can be appropriately set as long as the target reaction can proceed.
- conditions of the enzymatic method using aldolase are described in the production method (1-1) of the present invention. It may be the same as the above.
- Trp tryptophan
- IPA IPA
- Examples of Trp include L-Trp, D-Trp, and a mixture of L-Trp and D-Trp.
- the deamination of Trp can be performed by an organic chemical method and an enzymatic method using a deaminase.
- IPA IPA by deamination of Trp by an organic chemical method.
- a method for example, a method in which tryptophan is used as a starting material and reacted with pyridine aldehyde in the presence of a proton acceptor dehydrating base (eg, Japanese Patent Publication No. 62-501912, International Publication No. 1987/000169).
- a method of subjecting to acid hydrolysis after a condensation reaction using indole and ethyl-3-bromopyruvate ester oxime as raw materials for example, see European Patent Application No. 421946).
- the term “deaminase” refers to an enzyme capable of producing IPA from Trp.
- the production of IPA from Trp is essentially the conversion of the amino group (—NH 2 ) of Trp to an oxy group ( ⁇ O). Therefore, an enzyme that catalyzes this reaction may be referred to as another name such as amino acid deaminase, aminotransferase, amino acid oxidase and the like.
- deaminase is intended to mean any enzyme capable of producing IPA from Trp, and is an alias enzyme that catalyzes the reaction of producing IPA from Trp (eg, amino acid deaminase, aminotransferase, amino acid Oxidase) is also included in “deaminase”.
- an alias enzyme that catalyzes the reaction of producing IPA from Trp eg, amino acid deaminase, aminotransferase, amino acid Oxidase
- Examples of the method for producing IPA from Trp using amino acid deaminase or its producing bacterium include the method disclosed in International Publication No. 2009/0283338.
- the general formula of the reaction catalyzed by amino acid deaminase includes the following ⁇ formula: amino acid + H 2 O ⁇ 2-oxo acid + NH 3 >.
- Examples of the method for producing IPA from Trp using aminotransferase or its producing bacteria include, for example, East German Patent DD 297190, JP-A-59-95894, International Publication No. 2003/091396, US Patent Application Publication 2005. The method disclosed in the specification of / 0282260 is mentioned.
- a method for producing IPA from Trp using L-amino acid oxidase or its producing bacterium for example, US Pat. No. 5,002,963, John A. et al. Examples include those disclosed in Duerre et al. (Journal of Bacteriology 1975, vol 121, No. 2, p656-663), Japanese Patent Application Laid-Open No. 57-146573, International Publication No.
- the general formula of the reaction catalyzed by amino acid oxidase includes the following ⁇ formula: amino acid + O 2 + H 2 O ⁇ 2-oxo acid + H 2 O 2 + NH 3 >.
- a hydrogen peroxide-degrading enzyme such as catalase may be added to the reaction solution for the purpose of suppressing decomposition of the compound by hydrogen peroxide produced as a by-product.
- the reaction for generating IPA from Trp, the reaction for generating 4R-IHOG from IPA and pyruvic acid, and the reaction for generating 2S, 4R-monatin from 4R-IHOG may proceed separately or in parallel. These reactions may be carried out in one reaction tank. When performing these reactions in one reaction vessel, these reactions can be performed by adding the substrate and enzyme sequentially or simultaneously.
- a reaction in which Trp is deaminated by an enzymatic method using deaminase to generate IPA a reaction in which 4R-IHOG is generated from IPA and pyruvate by an enzymatic method using aldolase, and an L-amino acid amino acid
- 4R-monatin from 4R-IHOG is performed by transferase, (1) Trp and deaminase, (2) pyruvate and aldolase, and (3) L-amino acid and L-amino acid amino acid
- the transferase may be added sequentially or simultaneously in one reaction vessel.
- the contact with Trp and the deaminase is a deaminase (extract enzyme) extracted from Trp and the deaminase producing bacterium.
- a deaminase producing bacterium can be achieved by allowing a deaminase producing bacterium to coexist in a reaction solution (eg, culture solution).
- a reaction solution eg, culture solution.
- the deaminase-producing bacterium include a bacterium that naturally produces a deaminase and a transformant that expresses the deaminase.
- the pTB2 strain E.
- coli recombinant strain into which an amino acid deaminase gene derived from Providencia rettgeri strain is introduced) described in International Publication No. 2009/0283338 and Example 2 may be used.
- a functional promoter eg, phoA, phoC, trp, lac, tac promoter
- a deaminase expression plasmid may be introduced into a host in which a specific gene, for example, the aspC gene has been deleted.
- Examples of the extracted enzyme include purified enzyme, crude enzyme, immobilized enzyme, culture broth, and culture broth-treated product (eg, deaminase-containing fraction prepared from the above producing bacteria, crushed material and lysate of the above producing bacteria) Product).
- Examples of the treatment for obtaining a culture broth-treated product from the culture broth include heat treatment (42 to 80 ° C., pH 3 to 12, 1 minute to 24 hours), solvent treatment (eg, xylene, toluene, ethanol, isopropyl alcohol). , Surfactants (eg, Tween 20, Triton X-100), and lytic enzyme treatment (eg, lysozyme treatment).
- the temperature in this case is 4 ° C. to 60 ° C., preferably 20 ° C. to 37 ° C.
- the pH can be set to 3-12, preferably 7-9.
- the time can be set to about 5 minutes to 20 days, preferably about 1 hour to 7 days. Aeration stirring may or may not be performed while holding the broth.
- the deaminase producing bacterium may further express other enzymes (eg, aldolase, superoxide dismutase, L-amino acid aminotransferase, decarboxylase).
- Trp is preferably L-Trp. Trp may be added to the reaction solution in the form of a salt.
- the concentration of Trp in the reaction solution is, for example, 1 mM to 3M, preferably 20 mM to 1M, and more preferably 20 mM to 300 mM.
- deaminase, aldolase, L-amino acid aminotransferase, and oxaloacetate decarboxylase and / or 1 expressing them are used.
- Superoxide dismutase and / or transformants expressing it may also be used.
- These enzymes may be mutants.
- a transformant as described above can be used. Specifically, a transformant having an expression vector for the target gene in the cytoplasm, a transformant having the target gene introduced in the genome, and an expression vector for the target gene in the cytoplasm, And a transformant into which the target gene has been introduced.
- the expression vector used for the preparation of the transformant the expression vector as described above can be used.
- L-Trp when the production method (1-3) of the present invention is carried out in one reaction tank, L-Trp, L-Asp, PA, buffer (eg, phosphate buffer, Tris buffer) , A reaction solution containing PLP at a predetermined concentration can be used.
- concentration of L-Trp is, for example, 1 mM to 3M, preferably 10 mM to 1M, more preferably 50 mM to 300 mM.
- the L-Asp concentration is, for example, 1 mM to 3M, preferably 100 mM to 1M, more preferably 200 mM to 400 mM.
- L-Asp may be in the form of a salt (eg, sodium salt, potassium salt) or in a free form.
- the pH may be adjusted as appropriate after being added to the reaction solution.
- an alkaline solution eg, NaOH aqueous solution, KOH aqueous solution
- the PA concentration is, for example, 1 mM to 3M, preferably 10 mM to 100 mM.
- PA may be in the form of a salt (eg, sodium salt, potassium salt) or in a free form.
- the pH may be appropriately adjusted after charging into the reaction solution.
- the PLP concentration is, for example, 1 ⁇ M to 100 mM, preferably 10 ⁇ M to 1 mM.
- the reaction solution may further contain magnesium, phosphoric acid, and an antifoaming agent.
- magnesium used as a salt
- the form of the magnesium salt is not particularly limited, and examples thereof include magnesium chloride and magnesium sulfate.
- the magnesium concentration is, for example, 0.1 mM to 100 mM, preferably 0.5 mM to 5 mM.
- phosphoric acid used as a salt
- the form of the phosphate is not particularly limited.
- potassium salt eg, 1 potassium salt, 2 potassium salt, 3 potassium salt
- sodium salt eg, Monosodium salt, disodium salt, trisodium salt.
- the phosphoric acid concentration is, for example, 1 mM to 100 mM, preferably 10 mM to 50 mM.
- the concentration of the antifoaming agent is not particularly limited, but is, for example, 0.0001% to 1% (v / v), preferably 0.001% to 0.1% (v / v).
- Reaction conditions such as pH, temperature, aeration, stirring, and time can be appropriately set.
- the pH of the reaction solution is, for example, 5 to 10, preferably 6 to 9, and more preferably 7 to 8. Control of pH during the reaction can be achieved by appropriately adding an acid or an alkali.
- the acid or alkali used in this case is not particularly limited, and examples thereof include hydrochloric acid, phosphoric acid, sulfuric acid, ammonia gas, aqueous ammonia, aqueous NaOH solution, and aqueous KOH solution.
- the concentration of acid and alkali used for pH adjustment is not particularly limited. For example, when a solution is used, it is 0.1N to 20N, preferably 3N to 12N.
- the reaction temperature is, for example, 10 ° C. to 50 ° C., preferably 20 ° C. to 40 ° C., more preferably 25 ° C. to 35 ° C.
- the dissolved oxygen concentration in the reaction solution can be set by controlling the aeration / stirring conditions.
- a person skilled in the art can set the aeration and stirring conditions according to the container used.
- the aeration condition is, for example, 1/200 to 1 vvm, preferably 1/100 to 1/10 vvm
- the stirring condition is, for example, 100 rpm to 1000 rpm, preferably 400 rpm to 700 rpm. .
- Examples of the enzyme to be added to the reaction include purified enzyme, enzyme-expressing microbial cells, enzyme-expressed microbial cells, culture broth containing enzyme-expressing microbial cells, and culture broth treated with enzyme-expressing microbial cells.
- Examples of the treatment for obtaining a culture broth treated product from the culture broth include heat treatment (eg, 42 ° C. to 80 ° C., pH 3 to 12, 1 minute to 24 hours), solvent treatment (eg, xylene, toluene, ethanol, isopropyl). Alcohol), surfactant (eg, Tween 20, Triton X-100), and lytic enzyme treatment (eg, lysozyme treatment).
- the temperature of the culture broth is, for example, 4 ° C. to 60 ° C., preferably 20 ° C. to 37 ° C.
- the pH of the culture broth is, for example, 3 to 12, preferably 7 to 9.
- the holding time is, for example, about 5 minutes to 20 days, preferably about 1 hour to 7 days. While holding the culture broth, aeration and agitation may or may not be performed.
- the amount of each enzyme added to the reaction solution can be appropriately set by measuring each enzyme activity value in advance.
- Deaminase activity, aldolase activity, L-amino acid aminotransferase activity, and oxaloacetate decarboxylase activity can be measured, for example, by the following method.
- Deaminase activity 10 mM L-Phe, 100 mM NH 4 Cl, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, phenylalanine dehydrogenase (manufactured by Unitika Ltd., Thermoactinomyces intermedius), 25 ° C.
- the activity is calculated from the decrease of 340 nm.
- L-amino acid aminotransferase activity (L-Asp / ⁇ -KG activity): 100 mM L-Asp-Na-1aq, 10 mM ⁇ -KG-2Na, 50 ⁇ M PLP, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, MDH 2U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm. MDH used Malic Dehydrogenase from porcine heart (Sigma).
- Aldolase activity 2 mM 4-phenyl-4-hydroxy-2-oxo glutarate (PHOG), 100 mM Tris-HCl (pH 7.0), 1 mM MgCl 2 , 0.25 mM NADH, 10 U / ml lactate dehydrogenase (produced by Oriental Yeast Co., Ltd.) From Leuconostoc mesenteroides), 25 ° C.
- the activity is calculated from the decrease of 340 nm.
- Oxaloacetate decarboxylase activity 1 mM oxaloacetate, 100 mM Tris-HCl (pH 8.0), 0.25 mM NADH, 10 U / ml lactate dehydrogenase (produced by Oriental Yeast Co., Ltd., Leuconostoc mesenteroides), 25 ° C. The activity is calculated from the decrease of 340 nm. Based on the enzyme activity thus determined, the amount of enzyme added to the reaction solution may be as follows. The amount of deaminase added to the reaction solution is, for example, 0.1 to 20 U / ml, preferably 0.5 to 2 U / ml.
- the amount of aldolase added to the reaction solution is, for example, 1 to 1000 U / ml, preferably 10 to 100 U / ml.
- the amount of L-amino acid aminotransferase added to the reaction solution is, for example, 1 to 1000 U / ml, preferably 10 to 100 U / ml.
- the amount of oxaloacetate decarboxylase added to the reaction solution is, for example, 0.01 U / ml or more, preferably 0.1 U / ml or more.
- Each substrate may be added to the reaction system by a batch method or a feed method.
- Enzymes, enzyme-expressing cells, enzyme-treated cells, culture broth, or culture broth-treated products may also be added to the reaction system by a batch method or a feed method.
- the reaction time is, for example, 2 to 100 hours, preferably 4 to 50 hours, more preferably 8 to 25 hours.
- the reaction solution may be sterilized under appropriate conditions (eg, temperature, pH, time).
- the 2S, 4R-monatin-containing reaction solution obtained by any of the production methods (1-1), (1-2) and (1-3) of the present invention is used for known column processing, crystallization processing, extraction processing, etc.
- purified 2S, 4R-monatin can be obtained.
- the purified 2S, 4R-monatin can be subjected to the production method (2) of 2R, 4R-monatin or a salt thereof.
- the 2S, 4R-monatin-containing reaction solution obtained by any one of the production methods (1-1), (1-2) and (1-3) of the present invention is used as it is to produce 2R, 4R-monatin or a salt thereof. It can use for method (2).
- the present invention provides a method (2) for producing 2R, 4R-monatin or a salt thereof.
- the production method of the present invention comprises producing 2S, 4R-monatin or a salt thereof by the production method (1) of the present invention and isomerizing 2S, 4R-monatin or a salt thereof to produce 2R, 4R-monatin or a salt thereof. Producing salt.
- the isomerization of 2S, 4R-monatin to 2R, 4R-monatin can be performed by any method that allows the isomerization (eg, WO 2005/082850, WO 03/059865). See). However, from the viewpoint of improving the yield of 2R, 4R-monatin, the isomerization of 2S, 4R-monatin is preferably carried out by epicrystallization (see, for example, WO 2005/082850). ). Epicrystallization is a method in which isomerization and crystallization are performed simultaneously. In the present case, the isomerization reaction at the 2-position for converting 2S, 4R-monatin to 2R, 4R-monatin and the crystallization of the converted 2R, 4R-monatin are simultaneously performed by epicrystallization.
- the isomerization reaction may be performed in the presence of an aldehyde.
- aldehyde examples include aliphatic aldehydes and aromatic aldehydes, and aromatic aldehydes are preferable.
- 2S, 4R-monatin used in the isomerization reaction purified 2S, 4R-monatin may be used, or a reaction solution containing 2S, 4R-monatin may be used.
- Examples of aliphatic aldehydes are saturated with 1 to 7 carbon atoms such as formaldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, 1-butyraldehyde, n-barrel aldehyde, capronaldehyde, n-heptylaldehyde, acrolein, methacrolein, etc.
- an unsaturated aldehyde can be used.
- aromatic aldehydes examples include benzaldehyde, salicylaldehyde, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, o-nitrobenzaldehyde, p-nitrobenzaldehyde, 5-nitrosalicylaldehyde, 3,5-dichlorosalicylaldehyde, anisaldehyde, o -Vanillin, vanillin, furfural, pyridoxal, 5-phosphate pyridoxal and the like can be used.
- aromatic aldehyde, pyridoxal, 5-nitrosalicylaldehyde, and 3,5-dichlorosalicylaldehyde are particularly preferable.
- Aldehydes can be used in the range of 0.01 to 1 molar equivalent, more preferably 0.05 to 0.5 molar equivalent, relative to monatin present in the system.
- Epi crystallization is performed in the presence of an aldehyde, and a mixed solvent of water and an organic solvent is used as a solvent.
- a mixed solvent of water and an organic solvent is used as the organic solvent.
- an organic solvent miscible with water is used, but alcohols such as methanol, ethanol, propanol, and isopropanol are particularly preferable. Two or more different organic solvents may be mixed and used.
- the temperature of epicrystallization is preferably set in the range of 0 to 100 ° C., more preferably 40 to 80 ° C.
- the time for performing the epicrystallization is preferably set in the range of 10 hours to 1 week, more preferably 15 hours to 96 hours.
- PH is set in the range of 4 to 13, preferably 4.5 to 10, and more preferably 5 to 9. Adjustment of pH can be performed using an acid and an alkali.
- the acid used is not particularly limited, and an organic acid such as acetic acid or an inorganic acid such as hydrochloric acid or sulfuric acid can be used.
- the alkali is not particularly limited, and alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and organic bases such as ammonia and amines can be used.
- Each compound obtained by the above method is chromatographed using a known separation and purification means, for example, concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, phase transfer, activated carbon treatment, ion exchange resin or synthetic adsorption resin. Etc. can be isolated and purified by combining treatments as necessary.
- the compound used in the method of the present invention and the salt of the compound (target compound) produced by the method of the present invention are produced according to a method known per se, for example, by adding an inorganic acid or an organic acid to the target compound. Can do. Further, the target compound or a salt thereof may be a hydrate, and both a hydrate and a non-hydrate are included in the scope of the present invention.
- the compound (eg, Trp, IPA, 4R-IHOG, 2S, 4R-monatin) used in the production method of the present invention may be in the form of various salts such as sodium salt, potassium salt and ammonium salt.
- the compounds obtained by the production method of the present invention eg, IPA, 4R-IHOG, 2S, 4R-monatin, 2R, 4R-monatin
- HPLC analysis conditions In Examples 1 to 7, when HPLC analysis was performed, the HPLC analysis was performed under the conditions shown in the example. On the other hand, in Examples 8 to 15, HPLC analysis was performed under the following conditions.
- Detector UV absorption photometer (measurement wavelength: 210 nm)
- Gradient program see Table 1 below
- Example 1 Production of 2S, 4R-monatin from 4R-IHOG using Bacillus altitudinis AJ1616 cell extract CM2G agar medium (yeast extract 10 g / l, polypeptone 10 g / l, glucose 5 g / l, sodium chloride 5 g / l Bacillus altitudinis AJ1616 was applied to agar (15 g / l, pH 7.0) and cultured at 30 ° C. for 2 days.
- Bacillus altitudinis AJ161616 cell extract CM2G agar medium yeast extract 10 g / l, polypeptone 10 g / l, glucose 5 g / l, sodium chloride 5 g / l
- Bacillus altitudinis AJ161616 was applied to agar (15 g / l, pH 7.0) and cultured at 30 ° C. for 2 days.
- Enzyme production medium (yeast extract 10 g / l, polypeptone 10 g / l, glucose 1 g / l, dipotassium hydrogen phosphate 3 g / l, potassium dihydrogen phosphate 1 g / l, magnesium sulfate heptahydrate
- One platinum loop was inoculated into 3 ml of 0.1 g / l, ammonium sulfate 5 g / l), and cultured with shaking in a test tube at 30 ° C. for 16 hours. Bacteria were collected from 2 ml of the culture solution by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), and 1 ml of the cell suspension was prepared.
- Example 2 Purification of aminotransferase derived from Bacillus alterdinis AJ1616
- Purification of aminotransferase that produces 2S, 4R-monatin from the soluble fraction of Bacillus alterdinis AJ1616 was performed as follows. In the same manner as in Example 1, 2S, 4R-monatin synthesis reaction and 2S, 4R-monatin were quantified.
- CM2G agar medium yeast extract 10 g / l, polypeptone 10 g / l, glucose 5 g / l, sodium chloride 5 g / l, agar 15 g / l, pH 7.0. And cultured at 30 ° C. for 2 days. The obtained bacterial cells were inoculated into 160 ml of TB (Terrific Broth) medium, and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 16 hours.
- TB Target Broth
- the cells were collected from about 2000 ml of the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6) and 100 mM NaCl, and sonicated at 4 ° C. for 30 minutes. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- Example 3 Determination of N-terminal and internal amino acid sequences of aminotransferase derived from Bacillus altitudinis AJ1616
- the purified enzyme solution obtained in Example 2 was subjected to N-terminal amino acid sequence analysis. was gotten.
- the sample in the SDS-PAGE gel was trypsinized (pH 8.0, 35 ° C., 20 hours), and subjected to reverse phase HPLC to separate fragment peptides.
- an internal amino acid sequence of QLDSLSMGMLDVV SEQ ID NO: 5
- Both N-terminal amino acid sequence and internal amino acid sequence showed high homology with Bacillus pumilus SAFR-032-derived aminotransferase (YP — 001487343).
- Example 4 Cloning of Bacillus altitudinis AJ1616-derived aminotransferase gene Bacillus altitudinis AJ1616 was cultured in the same manner as in Example 1. Bacteria were collected from the obtained culture solution by centrifugation, and genomic DNA was extracted. A DNA fragment containing the aminotransferase gene was PCR amplified using the obtained genomic DNA as a template. Primer Bp-u300-f (5′-ctcagggaggcgaaaaaattattatt-3) was designed from the DNA sequence of 300 bp upstream and 200 bp downstream of the aminotransferase gene with reference to the genomic DNA sequence of Bacillus pumilus SAFR-032 (CP000813).
- Example 5 E.I. Expression of Bacillus altitudinis AJ1616-derived aminotransferase in E. coli (1) Construction of Bacillus altitudinis AJ1616-derived aminotransferase expression plasmid Bacillus altitudinis AJ1616-derived amino acid fragment Amplified using the genomic DNA of Bacillus altitudinis AJ1616 as a template.
- primer 1616AT-Nde-f (5′-ggaattccatATGAGCGGTTTTACAGCGTT-3 ′: SEQ ID NO: 8) and primer 1616-xho-r (5′-gtcaaggagtttttctgagTACCGTGTGTGCTCGATTGAC-3: SEQ ID NO: 8).
- the NdeI sequence in the aminotransferase gene uses the primer 1616-delNde-f (5′-GGATTGAAGGAACAcATGAAAAAAGCATGC-3 ′: SEQ ID NO: 10) and the primer 1616-delNde-r (5′-GCATGCTTTTCATCATCATTGTTTCCTTCATCC-3 ′): SEQ ID NO: 11 And converted.
- PCR was performed using KOD-plus-ver. 2 (Toyobo) was used under the following conditions.
- the obtained DNA fragment of about 1300 bp was subjected to restriction enzyme treatment with NdeI and XhoI and similarly ligated with pET-22b (Novagen) treated with NdeI and XhoI.
- pET-22b Novagen
- the target plasmid was extracted from the ampicillin resistant strain, and this plasmid was named pET-22-1616AT-His.
- Bacillus altitudinis AJ1616-derived aminotransferase (1616AT-His) with His-tag added at the C-terminus is expressed.
- E.E. 1616AT-His purification from E. coli expression strain The constructed expression plasmid pET-22-1616AT-His was obtained from E. coli.
- E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 160 ml of Overnight Express Instant Medium (Novagen) containing ampicillin 100 mg / l, and shaken at 37 ° C. for 16 hours using a 500 ml Sakaguchi flask. .
- the cells were collected from about 1000 ml of the obtained culture solution by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 20 mM Imidozole, and sonicated at 4 ° C. for 30 minutes. did.
- the cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- the obtained soluble fraction was mixed with His-tag protein purification column HisPrep FF 16/10 (Pharmacia (GE Healthcare Biosciences), CV, equilibrated with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and 20 mM Imidazole.
- the concentrated solution was diluted with Tris-HCl (pH 7.6) 20 mM, NaCl 100 mM and equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 100 mM.
- the protein adsorbed at the flow rate was eluted. 2S, 4R-monatin producing activity was confirmed for each eluted fraction, and fractions in which 2S, 4R-monatin producing activity was observed were collected and concentrated using Amicon Ultra-15 30k (Millipore). The concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to obtain a 1616AT-His solution.
- SpAld was prepared by the following method.
- a DNA fragment containing the SpAld gene was PCR-amplified using the plasmid DNA and ptrpSpALD described in JP-A-2006-204285 and Example 5 as a template.
- Primers SpAld-f-NdeI (5′-GGAATTCCATATACCACCAGACGCGCTCCAA-3 ′: SEQ ID NO: 12) and primer SpAld-r-HindIII (5′-GCCCCAAGCTTTCAGTACCCCGCCATTTCGC-3 ′: SEQ ID NO: 13) were used.
- coli rare codons (6L-ctc, 13L-ctc, 18P-ccc, 38P-ccc, 50P-ccc, 77P-ccc, 81P-ccc, 84R-cga) are 6L-ctg, 13L-ctg, 18P-ccg, Conversion was made to 38P-ccg, 50P-ccg, 77P-ccg, 81P-ccg, 84R-cgc.
- primer 6L-f (5′-ACCCACAGCCGCGCTGAACGGCATCATCCCG-3 ′: SEQ ID NO: 14) and primer 6L-r (5′-CGGATGATGCCGTTCAGGCGCGTCTGGGGT-3 ′: SEQ ID NO: 15) were used.
- primer 13L-f (5′-ATCATCCGCGCTCTCTGGAAGCCGGCAAGCC-3 ′: SEQ ID NO: 16) and primer 13L-r (5′-GGCTTGCCGGCTTCCCAGAGCGCGGATGATAT-3 ′: SEQ ID NO: 17) were used.
- primer 18P-f (5′-GAAGCCGGCAAGCCGGCTTTCACCGCTT-3 ′: SEQ ID NO: 18) and primer 18P-r (5′-AAGCAGGGTGAAAGCCCGCTTGCCCGCTCTC-3 ′: SEQ ID NO: 19) were used.
- primer 38P-f (5′-CTGACCGATGCCCCGTTAGACGGGCGTGGT-3 ′: SEQ ID NO: 20) and primer 38P-r (5′-ACCACGCCCGTCATACGGGGCATCGGTCAG-3 ′: SEQ ID NO: 21) were used.
- primer 50P-f (5′-ATGGAGCACAACCCGTACGATGTGCGCGGC-3 ′: SEQ ID NO: 22) and primer 50P-r (5′-GCCGCGCATCATCGTACGGGTGTGCTCCAT-3 ′: SEQ ID NO: 23) were used.
- primer 77P-81P, 84R the primer 77P-81P-84R-f (5'-CGGTCGCGCCGTCGGTCCACCCCGATCCGCGCGCATCCGCGCGCGCGCGCGCGGTCGCGCGGTGCG : SEQ ID NO: 25) was used.
- PCR was performed using KOD-plus (Toyobo) under the following conditions. 1 cycle 94 ° C, 2 min 25 cycles 94 ° C, 15 sec 55 ° C, 15 sec 68 °C, 60sec 1 cycle 68 ° C, 60 sec 4 °C
- the obtained DNA fragment of about 900 bp was subjected to restriction enzyme treatment with NdeI and HindIII, and ligated with pSFN Sm_Aet (International Publication No. 2006/0775486, Examples 1, 6, 12) similarly treated with NdeI and HindIII.
- pSFN Sm_Aet International Publication No. 2006/0775486, Examples 1, 6, 12
- this ligation solution E. coli JM109 was transformed, the target plasmid was extracted from the ampicillin resistant strain, and this plasmid was named pSFN-SpAld.
- One platinum loop of E. coli JM109 / pSFN-SpAld was inoculated into 50 ml of LB liquid medium containing 100 mg / l of ampicillin and shaken at 36 ° C. for 8 hours using a 500 ml Sakaguchi flask.
- 0.0006 ml of the obtained culture broth was used as a seed liquid medium containing 100 mg / l ampicillin (glucose 10 g, ammonium sulfate 5 g, potassium dihydrogen phosphate 1.4 g, soybean hydrolyzate 0.45 g as a nitrogen amount, sulfuric acid
- ampicillin glucose 10 g, ammonium sulfate 5 g, potassium dihydrogen phosphate 1.4 g, soybean hydrolyzate 0.45 g as a nitrogen amount, sulfuric acid
- the seed culture was performed at 33 ° C., aeration 1/1 vvm, stirring 700 rpm, pH was adjusted to 6.3 with ammonia until glucose was consumed.
- 15 ml of the culture broth thus obtained was added to a main liquid medium containing 100 mg / l of ampicillin (glucose 15 g, ammonium sulfate 5 g, phosphoric acid 3.5 g, soybean hydrolysate nitrogen amount 0.45 g, magnesium sulfate heptahydrate 1 g, iron (II) sulfate heptahydrate 0.05 g, manganese sulfate (II) pentahydrate 0.05 g, thiamine hydrochloride 1 mg, Dis home GD-113K (Nippon Yushi Co., Ltd.) 0.1 ml, pH 6 .3, 0.95 L with water)
- the mixture was added to a 1000 ml jar fermenter containing 285 ml, and main culture was started.
- Fractions having aldolase activity were collected and concentrated using Amicon Ultra-15 10k (Millipore). The obtained concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to obtain a SpAld solution.
- the aldolase activity the aldol degradation activity using PHOG as a substrate was measured under the following conditions. Reaction conditions: Phosphate buffer (pH 7.0) 50 mM, PHOG 2 mM, NADH 0.25 mM, MgCl 2 1 mM, lactate dehydrogenase 16 U / ml, 25 ° C., absorbance at 340 nm was measured.
- the pTB2 strain was prepared by the following method.
- One platinum loop of the pTB2 strain described in International Publication No. 2009/0283338 and Example 2 was inoculated into 50 ml of a TB liquid medium containing 100 mg / l of ampicillin and shaken at 37 ° C. for 16 hours using a 500 ml Sakaguchi flask.
- the obtained culture broth was used as the Sakaguchi flask culture solution (TB medium) of the pTB2 strain.
- Example 7 2S, 4R-monatin synthesis by 2S, 4R-monatin producing active bacteria (1) 2S, 4R-monatin synthesis by bacteria Nutrient broth (NB) agar medium or CM2G agar medium (yeast extract 10 g / l, Polypeptone 10 g / l, Glucose 5 g / l, NaCl 5 g / l, Agar 15 g / l, pH 7.0), Rhizobium radiobacter LAT1, Rhizobium radiobacter AJ11568, Dietzia maris spo AJ27. AJ3447, Stenotrophomonas sp.
- NB Nutrient broth
- CM2G agar medium
- Rhizobium radiobacter LAT1 Rhizobium radiobacter AJ11568
- Dietzia maris spo AJ27. AJ3447 Stenotrophomonas sp.
- AJ13127 Pseudomonas chlororaphis subsp. Chlororaphis NBRC3904, Micrococcus luteus NBRC3067, Stenotrophomonas sp. AJ11634, Pseudomonas putida NBRC12668, Ocrobactrum pseudoligonense AJ3735, Stenotrophomonas sp. AJ1591, Stenotrophomonas sp. AJ3839, Brevundimonas diminuta AJ3958, Pseudomonas citronocllolis ATCC 13694, Arthrobacter sp. IAM1390, Rhizobium sp.
- Rhizobium radiobacter AJ2777 Rhizobium radiobacter AJ2777, Burkholderia sp. AJ3084, Microbacterium sp. AJ2787, Pseudomonas taetrolens ATCC4683, Rhizobium radiobacter ATCC4452, Rhizobium radiobacter AJ2557, Carnimonas sp.
- Rhizobium radiobacter NBRC12667 Rhizobium radiobacter NBRC12667, Pseudomonas fragagi NBRC3458, Rhizobium radiobacter NBRC12664, Corynebacterium ammonianes NBRC12072m AJ1594, Rhizobium radiobacter ATCC 6466, Pseudomonas synxantha NBRC3912, Rhizobium radiobacter ATCC 4720, or Pseudomonas sp. LMG2833 was applied and cultured at 30 ° C. for 2 days.
- Enzyme production medium (yeast extract 10 g / l, polypeptone 10 g / l, glucose 1 g / l, dipotassium hydrogen phosphate 3 g / l, potassium dihydrogen phosphate 1 g / l, magnesium sulfate heptahydrate
- One platinum loop was inoculated into 3 ml of 0.1 g / l, ammonium sulfate 5 g / l), and cultured with shaking in a test tube at 30 ° C. for 16 hours. Bacteria were collected from 2 ml of the culture solution by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), and 1 ml of the cell suspension was prepared.
- yeast extract 10 g / l, polypeptone 20 g / l, glucose 20 g / l, agar 15 g / l) Lypolytica NBRC0746 was applied and cultured at 30 ° C. for 2 days.
- the obtained cells were inoculated into 3 ml of YPD medium (yeast extract 10 g / l, polypeptone 20 g / l, glucose 20 g / l), and cultured with shaking in a test tube at 30 ° C. for 16 hours.
- the mother liquor was cooled to 10 ° C. and then stirred for 5 hours to crystallize 2S, 4R-monatin potassium salt dihydrate.
- the precipitated crystals were separated by filtration (wet crystals 32.74 g) and dried under reduced pressure to obtain 9.88 g (15.68 mmol) of the desired 2S, 4R-monatin potassium salt dihydrate (HPLC purity). : 55.5%).
- the water content and potassium content of the obtained crystals (2S, 4R-monatin potassium salt dihydrate) were analyzed by moisture measurement and cation analysis by ion chromatography. Details of the water measurement method and the cation analysis method performed are shown below.
- Example 9 Isomerization using 5-nitrosalicylaldehyde 0.15 g (0.38 mmol) of 2S, 4R-monatin potassium salt dihydrate was added to 10.0 g of a 70% aqueous ethanol solution at 60 ° C. And completely dissolved. To the solution, 7.6 mg (0.045 mmol) of 5-nitrosalicylaldehyde and 7.5 ⁇ L (0.13 mmol) of acetic acid were added and stirred at 60 ° C. for 48 hours. When the reaction solution was analyzed by HPLC and quantified, the molar ratio of 2S, 4R-monatin and 2R, 4R-monatin in the reaction solution was 1: 2.1.
- Example 10 Isomerization reaction using pyridoxal hydrochloride 0.15 g (0.38 mmol) of 2S, 4R-monatin potassium salt dihydrate was added to 10.0 g of a 70% aqueous ethanol solution at 60 ° C. It was completely dissolved. To the solution, 9.1 mg (0.045 mmol) of pyridoxal hydrochloride and 7.5 ⁇ L (0.13 mmol) of acetic acid were added and stirred at 60 ° C. for 48 hours. When the reaction solution was analyzed by HPLC and quantified, the molar ratio of 2S, 4R-monatin and 2R, 4R-monatin in the reaction solution was 1: 1.3.
- Example 11 Isomerization with 5-phosphate pyridoxal monohydrate 0.15 g (0.38 mmol) of 2S, 4R-monatin potassium salt dihydrate was added to 10.0 g of 70% aqueous ethanol. And completely dissolved at 60 ° C. To the solution, 12.8 mg (0.048 mmol) of pyridoxal 5-phosphate monohydrate and 7.5 ⁇ L (0.13 mmol) of acetic acid were added and stirred at 60 ° C. for 48 hours. When the reaction solution was analyzed by HPLC and quantified, the molar ratio of 2S, 4R-monatin and 2R, 4R-monatin in the reaction solution was 1: 1.1.
- Example 12 Isomerization reaction using salicylaldehyde 0.15 g (0.38 mmol) of 2S, 4R-monatin potassium salt dihydrate was added to 10.0 g of a 70% aqueous ethanol solution, and completely at 60 ° C. Dissolved in. To the solution, 5.3 mg (4.6 ⁇ L, 0.043 mmol) of salicylaldehyde and 7.5 ⁇ L (0.13 mmol) of acetic acid were added and stirred at 60 ° C. for 48 hours. When the reaction solution was analyzed by HPLC and quantified, the molar ratio of 2S, 4R-monatin and 2R, 4R-monatin in the reaction solution was 1: 0.6.
- Example 13 Isomerization reaction using 3,5-dichlorosalicylaldehyde 0.15 g (0.38 mmol) of 2S, 4R-monatin potassium salt dihydrate was added to 10.0 g of 70% aqueous ethanol solution, It was completely dissolved at 60 ° C. To the solution, 8.1 mg (0.042 mmol) of 3,5-dichlorosalicylaldehyde and 7.5 ⁇ L (0.13 mmol) of acetic acid were added and stirred at 60 ° C. for 48 hours. When the reaction solution was analyzed by HPLC and quantified, the molar ratio of 2S, 4R-monatin and 2R, 4R-monatin in the reaction solution was 1: 1.5.
- Example 14 Production of 2R, 4R-monatin potassium salt monohydrate by isomerization crystallization using 2S, 4R-monatin potassium salt dihydrate as a starting material 2S, 4R-monatin potassium salt dihydrate
- the Japanese product is added to a 20% aqueous ethanol solution and completely dissolved at 60 ° C.
- 5-nitrosalicylaldehyde is added at 5 mol% with respect to 2S, 4R-monatin
- acetic acid is added at 30 mol% with respect to 2S, 4R-monatin, followed by stirring for 48 hours.
- Example 15 Isomerization reaction using glyoxylic acid 0.15 g (0.38 mmol) of 2S, 4R-monatin potassium salt dihydrate was added to 10.0 g of 70% aqueous ethanol solution, and completely at 60 ° C. It was dissolved in. To the solution, 5.1 mg (0.069 mmol) of glyoxylic acid and 7.5 ⁇ L (0.13 mmol) of acetic acid were added and stirred at 60 ° C. for 48 hours. When the reaction solution was analyzed by HPLC and quantified, the molar ratio of 2S, 4R-monatin and 2R, 4R-monatin in the reaction solution was 1: 0.07.
- Example 16 Preparation of mutants of L-amino acid aminotransferase (LAT) derived from AJ1616 strain and measurement of specific activity against various keto acids (1) Preparation of mutant LAT expression plasmid by site-directed mutation Mutant form by site-specific mutation The AJ1616 strain-derived LAT expression plasmid was prepared according to the protocol of the QuikChange Site-Directed Mutagenesis Kit manufactured by Stratagene. A set of DNA primers designed to introduce a mutation (substitution) of the target nucleotide residue and to be complementary to each strand of the double-stranded DNA was synthesized. The nucleotide sequences of the prepared mutants and the primers used for the preparation are shown in Tables 5 and 6, respectively.
- a mutant plasmid was prepared under the following PCR conditions. 1 cycle 95 ° C 1 min 18 cycles 95 ° C 30 sec 55 ° C 1 min 68 °C 8 min 4 ° C after the end of cycles
- ID136 is a double mutant of S258G / I289A, and was constructed by repeating the same procedure using I289A mutation-introducing primers after preparing the S258G mutant plasmid.
- ID189 is a K39R / T288G double mutant, and was constructed by repeating the same procedure using a primer for K39R mutation introduction after preparing an ID166 (T288G) mutant plasmid.
- ID296 is a double mutant of Q287E / T288G, but was constructed by repeating the same operation using a primer for introducing a mutation of Q287E / T288G after preparing a T288G mutant plasmid.
- mutant AJ1616LAT expression plasmid was used to express E. coli.
- E. coli JM109 (DE3) was transformed to produce a mutant AJ1616LAT expression strain. Mutant AJ1616LAT expression strain grown on LB-amp (100 mg / l) plate, pET22-AJ1616LATmut-His (C) / E.
- the cells of E. coli JM109 (DE3) were inoculated into 100 ml of Overnight Express TB Medium (Novagen) containing ampicillin 100 mg / l, and shake-cultured at 37 ° C. for 16 hours using a Sakaguchi flask.
- bacterial cells were collected from the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic crushing. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction. The obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidazole 10 mM, and was adsorbed on the carrier.
- L-Asp / ⁇ -KG ( ⁇ -ketoglutarate) activity L-Asp-Na 100 mM, ⁇ -KG-2Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH 2U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm. MDH used Malic Dehydrogenase from porcine heart (Sigma). The L-Asp / ⁇ -KG activity is shown in the item “ ⁇ -KG” of aminotransferase activity in Table 9.
- L-Asp / PA activity L-Asp-Na 100 mM, PA-Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH (same as above) 2 U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm.
- the L-Asp / PA activity is shown in the item “PA” of aminotransferase activity in Table 9.
- L-Asp / ( ⁇ ) -MHOG (4-hydroxy-4-methyl-2-ketoglutarate) activity: L-Asp-Na 100 mM, ( ⁇ ) -MHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM NADH 0.25 mM, MDH 2 U / ml, LDH 10 U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm.
- LDH D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used. LDH was added to remove PA mixed in a small amount in ( ⁇ ) -MHOG.
- the L-Asp / ( ⁇ ) -MHOG activity is shown in the item “ ⁇ MHOG” of aminotransferase activity in Table 9.
- L-Asp / 4R-IHOG and L-Asp / IPA activity measurement The 2S, 4R-monatin producing activity from 4R-IHOG, which is the target activity, and the L-Trp by-living property from IPA were measured.
- As the amino donor substrate for transamination reaction 100 mM L-Asp was used, transamination reaction was performed on 10 mM of various keto acids, and the amount of amino acid produced was determined by UPLC or HPLC, and the specific activity was calculated.
- L-Asp / 4R-IHOG activity (10 mM): L-Asp-Na 100 mM, 4R-IHOG 10 mM (including a trace amount of 4S-IHOG), PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, 25 ° C. UPLC analysis was performed, and the produced 2S, 4R-monatin and 2S, 4S-monatin were quantified. As the reaction stop solution, a 200 mM Na citrate solution (pH 4.5) was used. The L-Asp / 4R-IHOG activity is shown in the item “4R-IHOG” of aminotransferase activity in Table 9.
- L-Asp / IPA activity L-Asp-Na 100 mM, IPA 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM (pH adjusted to 8.0 with 1N NaOH after preparation of reaction solution), 25 ° C.
- UPLC analysis was performed and the produced Trp was quantified.
- As the reaction stop solution a 200 mM Na citrate solution (pH 4.5) was used.
- the L-Asp / IPA activity is shown in the item “IPA” of aminotransferase activity in Table 9.
- the produced monatin and Trp were quantified using an ACQUITY UPLC system manufactured by Waters. The measurement conditions are shown below.
- 2S, 4R-monatin can be fractionally quantified to 1.1 min
- 2S, 4S-monatin can be fractionated to 1.5 min
- Trp can be fractionated to 1.3 min.
- HPLC conditions (monatin, Trp, IPA, IAA (indole acetic acid), IAD (indole aldehyde) quantitative conditions)
- Example 17 E.I. Construction of E. coli JM109 ⁇ aspC strain and production of deaminase expression broth E. coli JM109 ⁇ aspC was constructed by the following method.
- E. E. coli JM109 / pKD46 was cultured at 30 ° C. overnight on LB-amp (100 mg / l) plates.
- the obtained bacterial cells were inoculated into 50 ml of LB (containing 100 mg / l of Amp and 10 mM of L-arabinose). This was cultured with shaking at 30 ° C. using a Sakaguchi flask.
- Bacteria were collected from the obtained culture solution by centrifugation, washed with 10% Glycerol, centrifuged again and collected. This was suspended in 10% Glycerol to obtain a competent cell.
- the obtained PCR product was extracted from agarose and used as a DNA fragment for aspC gene disruption. PCR was performed using KOD-plus-ver. 2 (Toyobo). A competent cell was transformed with the purified DNA fragment, and the target transformant was selected on an LB-Cm (20 mg / l) plate at 37 ° C. It was confirmed by colony PCR that attL-cat-attR was inserted into the aspC gene region of the transformant.
- the primers used were the primer aspC-up (5'-AACCCTCTTGCACAGGGTAAAAAGCTGAAC-3 ': SEQ ID NO: 38), primer attL-1 (5'-TAGTGACCCTTTCGTTGC-3': SEQ ID NO: 39), primer aspC-down (5'-GCCTGTGCAGTGAGTGGTGG ⁇ 5 ′: SEQ ID NO: 40) and primer attR-1 (5′-TTACGTTTCTCGTTCAGC-3 ′: SEQ ID NO: 41).
- Z-taq (TAKARA) was used.
- the obtained transformant was inoculated into 3 ml of LB (Cm 20 mg / l) and cultured with shaking at 37 ° C. for 6 hours.
- Bacteria were collected from the obtained culture solution by centrifugation, washed with 10% Glycerol, centrifuged again and collected. This was suspended in 10% Glycerol to obtain a competent cell.
- the competent cell was transformed with pMW-intxis-ts. The desired transformant was selected on an LB-amp (100 mg / l) plate at 30 ° C. The obtained transformant was cultured overnight on an LB plate at 42 ° C., and then the cells were streaked on an LB-amp (100 mg / l) plate and an LB-Cm (20 mg / l) plate, respectively, at 37 ° C. Cultured.
- coli JM109 ⁇ aspC is a deaminase expression plasmid pTB2 and E. coli.
- E. coli JM109 ⁇ aspC was transformed and constructed. This strain was cultured overnight on an LB-amp (100 mg / l) plate at 37 ° C. The obtained cells were inoculated into 100 ml of TB-amp (100 mg / l), and cultured with shaking at 37 ° C. for 16 hours using a Sakaguchi flask. The obtained culture broth was used as Ps_aad broth.
- Example 18 Construction of an Oxaloacetate Decarboxylase Expression Strain Genoscript was requested to synthesize the gene for OAA Decarboxylase derived from Pseudomonas putida KT2440 strain, and a DNA fragment containing the OAA Decaboxylase gene was inserted into pUC57. The frequency of codon usage is E. coli. Optimized for expression in E. coli (see SEQ ID NOs: 42, 43). This plasmid was digested with NdeI and XhoI, inserted into pET22b digested with NdeI and XhoI, and the resulting plasmid was named pET22-PpODC-His (C).
- E. coli E. coli BL21 (DE3) was transformed into an expression strain pET22-PpODC-His (C) / E. coli BL21 (DE3) was obtained.
- PpODC-His (C) expression strain grown on LB-amp (100 mg / l) plate, pET22-PpODC-His (C) / E.
- the cells of E. coli BL21 (DE3) were inoculated into 100 ml of Overnight Express TB Medium (Novagen) containing 100 mg / l of ampicillin, and cultured with shaking in a Sakaguchi flask at 30 ° C. for 16 hours.
- bacterial cells were collected from the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic crushing. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction. The obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidazole 10 mM, and was adsorbed on the carrier.
- Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, LDH 10 U / ml, 25 ° C. Activity was calculated from the decrease in 340 nm.
- LDH D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used. Reaction and analysis were performed on a 1 ml scale, and the activity value at the dilution stage where the value of the measurement result (Sample ⁇ 340 nm / min) ⁇ (Blank ⁇ 340 nm / min) was in the range of 0.05 to 0.15 was adopted.
- Tris-HCl (pH 7.6) 20 mM, BSA 0.01% was used.
- Example 19 One-pot synthesis reaction of 2S, 4R-monatin from L-Trp 100 mM (WT, ID136, ID166) Using purified mutant AJ1616LAT, the reaction was carried out for 22 hours under the following conditions. The reaction was performed in 1 ml using a test tube. Sampling was performed after 14, 18, and 22 hours. The sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa, and the filtrate was analyzed. HPLC was used for the analysis.
- Reaction conditions L-Trp 100 mM, PA-Na 50 mM, L-Asp-Na 300 mM, MgCl 2 1 mM, PLP 50 ⁇ M, Tris-HCl 100 mM, KPB 20 mM, pH 7.0, Ps_aad broth 40%, SpAld purified enzyme 0.2 mg / Ml, OAA DCase commercial enzyme 10 U / ml, mutant AJ1616LAT purified enzyme 2 U / ml (vs 10 mM 4R-IHOG), SOD commercial enzyme 200 U / ml, 25 ° C., 140 rpm.
- Ps_aad broth prepared according to the method described in Example 17.
- SpAld purified enzyme SpALD-expressing strains were jar-cultured according to the method described in Example 6, followed by heat treatment at 60 ° C. for 1 hour. From 100 ml of the obtained culture solution after heat treatment, the cells were collected by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), and subjected to ultrasonic disruption. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- Non-adsorbed protein that was not adsorbed on the carrier was washed away using 20 mM of ammonium sulfate 1M, Tris-HCl (pH 7.6), and then the ammonium sulfate concentration was linearly changed from 1M to 0M and adsorbed at a flow rate of 8 ml / min. The protein was eluted. Fractions in which activity was detected were collected and concentrated using Amicon Ultra-15 10k (Millipore). The obtained concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to obtain a SpAld solution.
- LDH D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used.
- Mutant AJ1616LAT Mutant AJ1616LAT expression strain grown on LB-amp (100 mg / l) plate, pET22-AJ1616LATmut-His (C) / E. The cells of E.
- coli JM109 (DE3) were inoculated into 100 ml of Overnight Express TB Medium (Novagen) containing ampicillin 100 mg / l, and shake-cultured at 37 ° C. for 16 hours using a Sakaguchi flask. After completion of the culture, bacterial cells were collected from the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic crushing. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- the obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidazole 10 mM, and was adsorbed on the carrier.
- Tris-HCl pH 7.6
- the protein that was not adsorbed on the carrier was washed away with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, Imidazole 10 mM, and then Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, Imidazole 150 mM.
- the produced ID136, ID166 mutant enzyme showed an effect of improving 2S, 4R-monatin yield compared to the wild type (Table 10).
- Example 20 One-pot reaction of 2S, 4R-monatin from 100 mM Trp (ID166, 400 ml scale) Using purified AJ1616LAT-ID166, the reaction was carried out for 6 hours under the following conditions. The reaction was performed in 400 ml using a 1 L S-jar. Sampling was performed as appropriate, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa, and the filtrate was analyzed. Analysis was performed by HPLC and capillary electrophoresis.
- Reaction conditions L-Trp 100 mM, PA-Na 50 mM, L-Asp-Na 300 mM, MgCl 2 1 mM, PLP 50 ⁇ M, KPB (pH 7.6) 20 mM, pH ⁇ 7.6 (1 MH 2 SO 4 ), Ps_aad broth 40%, SpAld broth 10%, PpODC 5 U / ml, AJ1616LAT-ID166 4 U / ml (vs 10 mM 4R-IHOG), SOD 100 U / ml, 25 ° C., 500 rpm, Air 20 ml / min (1/20 vvm).
- Ps_aad broth is pTB2 / E. E. coli JM109 ⁇ aspC broth was used.
- the SpAld broth the heat-treated broth described in Example 19 was used.
- the purified enzyme described in Example 18 was used for PpODC.
- SOD Superoxide Dismutase from bovine liver (Sigma) was used.
- Example 21 2S, 4R-monatin one-pot reaction from 150 mM L-Trp (ID189, 80 ml scale) Using purified AJ1616LAT-ID189, the reaction was carried out for 27 hours under the following conditions. The reaction was carried out at 80 ml using a 250 mL S-jar. Sampling was performed as appropriate, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa, and the filtrate was analyzed. Analysis was performed by HPLC and capillary electrophoresis.
- coli JM109 ⁇ aspC broth was used.
- the SpAld broth the heat-treated broth described in Example 19 was used.
- the purified enzyme described in Example 18 was used for PpODC.
- SOD a superoxide dismount from bovine river (Sigma) was used.
- Example 22 Isolation of 2S, 4R-monatin To 435.66 g of the permeate obtained by treating 435.45 g (lot 101213 J4) of the enzyme reaction solution of Example 20 with UF (MWCO: 3000), .59 g was added and stirred at room temperature (about 26 ° C.) for 1 hour.
- the activated carbon was filtered with Kiriyama filter paper (5C), and the obtained filtrate was transferred to a 1 L four-necked flask. The flask was immersed in a 5 ° C. constant temperature bath, neutralized with 35% hydrochloric acid so as to have a pH of 3.5, and stirred with a mechanical stirrer (120 rpm).
- Example 23 Synthesis of 2R, 4R-monatin 3.10 g (10.4 mmol) of 2S, 4R-monatin obtained in Example 22 and 1.165 g (10.4 mmol) of 50% KOH were dissolved in 3.27 g of water. Further, 1.3 g of EtOH, 0.0869 g (0.052 mmol) of 5-nitrosalicylaldehyde and 0.187 g (3.12 mmol) of acetic acid were added. After 25 hours, 20.5 g of EtOH and 10 mg of seed crystals (2R, 4R-monatin) were added, and the mixture was further stirred for 46.5 hours.
- the obtained slurry solution was cooled to room temperature, filtered, washed with 4 g of 85% EtOH water, and then dried under reduced pressure at 40 ° C. to obtain 2.3 g of crude 2R, 4R-monatin. did. Dissolve 2.1 g of the obtained crude 2R, 4R-monatin in 6 mL of water, add 0.2 g of BA charcoal, stir at room temperature (around 25 ° C.) for 1 hour, and filter the treatment liquid with a 0.45 ⁇ m membrane filter. The filtrate was concentrated under reduced pressure to 6.38 g. EtOH 12g was dripped at the concentrate at 45 degreeC, and it stirred for 1 hour.
- Example 24 2S, 4R-monatin one-pot reaction from 150 mM L-Trp (ID 296, 80 ml scale) Using purified AJ1616LAT-ID296, the reaction was carried out for 51 hours under the following conditions. The reaction was performed at 80 ml using a 250 ml jar. Sampling was performed appropriately, the sample was diluted with TE buffer, ultrafiltration was performed using Amicon Ultra-0.5 mL centrifugal filter 10 kDa, and the filtrate was analyzed. HPLC was used for the analysis.
- E. coli JM109 ⁇ aspC broth was used.
- the SpAld broth the heat-treated broth described in Example 19 was used.
- the purified enzyme described in Example 18 was used for PpODC.
- SOD a superoxide dismount from bovine river (Sigma) was used.
- Example 25 Purification of Rhizobium radiobacter AJ3976-derived aminotransferase
- the aminotransferase producing 2S, 4R-monatin was purified from the soluble fraction of Rhizobium radiobacter AJ3976 as follows. L-Asp-Na-1aq 100 mM, 4R-IHOG 10 mM (including a trace amount of 4S-IHOG), PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, reacted at 25 ° C., and the produced 2S, 4R-monatin was Quantified by UPLC analysis.
- Rhizobium radiobacter AJ3976 was applied to an LB agar medium and cultured at 30 ° C for 2 days.
- Enzyme production medium yeast extract 10 g / l, tryptone 10 g / l, glucose 1 g / l, dipotassium hydrogen phosphate 3 g / l, potassium dihydrogen phosphate 1 g / l, magnesium sulfate heptahydrate
- Enzyme production medium yeast extract 10 g / l, tryptone 10 g / l, glucose 1 g / l, dipotassium hydrogen phosphate 3 g / l, potassium dihydrogen phosphate 1 g / l, magnesium sulfate heptahydrate
- One platinum loop was inoculated into 160 ml of 0.1 g / l, ammonium sulfate 5 g / l), and cultured with shaking in a 500 ml Sakaguchi flask at 30 °
- Bacterial cells were collected from about 1920 ml of the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), and sonicated at 4 ° C. for 30 minutes. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- Example 26 Determination of N-terminal amino acid sequence of aminotransferase derived from Rhizobium radiobacter AJ3976
- the purified enzyme solution obtained in Example 25 was subjected to N-terminal amino acid sequence analysis. It was.
- the N-terminal amino acid sequence is Agrobacterium tumefaciens str. It showed high homology with C58-derived aspartate aminotransferase (AAK87940).
- Example 27 Cloning of Rhizobium radiobacter AJ3976-derived aminotransferase gene Rhizobium radiobacter AJ3976 was cultured in the same manner as in Example 25. Bacteria were collected from the obtained culture solution by centrifugation, and genomic DNA was extracted. A DNA fragment containing the aminotransferase gene was PCR amplified using the obtained genomic DNA as a template. Primers were obtained from Agrobacterium tumefaciens str.
- the primer Ag-u100-f (5'-ctgggtcagatagccggctttttgacc-3 ': SEQ ID NO: 45) and the primer Ag- designed from the 100 bp upstream and 100 bp downstream DNA sequences of the aminotransferase gene d100-r (5′-ccactttcatcatgctgctgtttctcg-3 ′: SEQ ID NO: 46) was used.
- PCR was performed using KOD-plus-ver. 2 (Toyobo) was used under the following conditions. 1 cycle 94 ° C, 2 min 25 cycles 98 ° C, 10 sec 55 ° C, 10 sec 68 °C, 60sec 1 cycle 68 ° C, 60 sec 4 °C
- Example 28 E.M. Expression of Rhizobium radiobacter AJ3976-derived aminotransferase in E. coli
- Construction of Rhizobium radiobacter AJ3976-derived aminotransferase expression plasmid Rhizobium radiobacter AJ3976-derived Rhizobium radiobacter AJ3976-derived amino acid As the primer, primer 3976AT-Nde-f (5′-ggaattccatATGGCCTCTCCTGCCCGACATCTCT-3 ′: SEQ ID NO: 49) and primer 3976-xho-r (5′-actccgctcgagACGGCAGCGGCGCAGAAACGCTGA-3 ′: SEQ ID NO: 50) were used. PCR was performed using KOD-plus-ver.
- the obtained DNA fragment was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI.
- pET-22b Novagen
- the target plasmid was extracted from the ampicillin resistant strain, and this plasmid was named pET-22-3976AT-His.
- This plasmid expresses Rhizobium radiobacterium AJ3976-derived aminotransferase (3976AT-His) with His-tag added to the C-terminus.
- E.E. Purification of 3976AT-His from E. coli expression strain The constructed expression plasmid pET-22-3976AT-His was transformed into E. coli.
- E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 160 ml of Overnight Express TB Medium (Novagen) containing ampicillin 100 mg / l and shaken at 37 ° C. for 16 hours using a 500 ml Sakaguchi flask.
- the cells were collected from about 1000 ml of the obtained culture solution by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 20 mM Imidozole, and sonicated at 4 ° C. for 30 minutes. did.
- the cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- the obtained soluble fraction was mixed with His-tag protein purification column HisPrep FF 16/10 (Pharmacia (GE Healthcare Biosciences), CV, equilibrated with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and 20 mM Imidazole.
- Example 29 Specific activity measurement results of AJ3976LAT for various keto acids (1) Measurement of L-Asp / ⁇ -KG, L-Asp / PA, and L-Asp / ( ⁇ ) -MHOG activity by colorimetric method AJ3976LAT Activity measurements on various substrates were performed. As the amino donor substrate for the transamination reaction, 100 mM L-Asp was used, and the specific activity against 10 mM of various keto acids was measured by a colorimetric method.
- L-Asp / ⁇ -KG activity L-Asp-Na-1aq 100 mM, ⁇ -KG-2Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH 2 U / ml, 25 ° C. . The activity is calculated from the decrease of 340 nm. MDH used Malic Dehydrogenase from porcine heart (Sigma). The L-Asp / ⁇ -KG activity is shown in the item “ ⁇ -KG” of aminotransferase activity in Table 13.
- L-Asp / PA activity L-Asp-Na-1aq 100 mM, PA-Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH (same as above) 2 U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm.
- the L-Asp / PA activity is shown in the item “PA” of aminotransferase activity in Table 13.
- L-Asp / ( ⁇ ) -MHOG activity L-Asp-Na-1aq 100 mM, ( ⁇ ) -MHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH (same as above) ) 2U / ml, LDH 10U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm.
- LDH D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used. LDH was added to remove PA mixed in a small amount in ( ⁇ ) -MHOG.
- the L-Asp / ( ⁇ ) -MHOG activity is shown in the item “ ⁇ MHOG” of the aminotransferase activity in Table 13.
- L-Asp / 4R-IHOG, L-Asp / ( ⁇ ) -IHOG, and L-Asp / IPA activity measurement 2S, 4R-monatin producing activity from 4R-IHOG which is the target activity, ( ⁇ )- 2S, 4R-monatin and 2S, 4S-monatin producing activity from IHOG, and L-Trp side-life from IPA were measured, respectively.
- the amino donor substrate for the transamination reaction 100 mM L-Asp was used, transamination reaction was performed on 10 mM of various keto acids, the amount of amino acid produced was quantified by UPLC, and the specific activity was calculated.
- L-Asp / 4R-IHOG activity L-Asp-Na-1aq 100 mM, 4R-IHOG 10 mM (including a trace amount of 4S-IHOG), PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, 25 ° C. UPLC analysis was performed, and the produced 2S, 4R-monatin and 2S, 4S-monatin were quantified. As the reaction stop solution, a 200 mM Na citrate solution (pH 4.5) was used. The L-Asp / 4R-IHOG activity is shown in the item “4R-IHOG” of aminotransferase activity in Table 13.
- L-Asp / ( ⁇ ) -IHOG activity L-Asp-Na-1aq 100 mM, ( ⁇ ) -IHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, 25 ° C. UPLC analysis was performed, and the produced 2S, 4R-monatin and 2S, 4S-monatin were quantified. As the reaction stop solution, a 200 mM Na citrate solution (pH 4.5) was used. The L-Asp / 4R-IHOG activity is shown in the item “ ⁇ IHOG” of aminotransferase activity in Table 13.
- L-Asp / IPA activity L-Asp-Na-1aq 100 mM, IPA 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM (pH adjusted to 8.0 with 1N NaOH after preparation of reaction solution), 25 ° C. .
- UPLC analysis was performed and the produced Trp was quantified.
- As the reaction stop solution a 200 mM Na citrate solution (pH 4.5) was used.
- the L-Asp / IPA activity is shown in the item “IPA” of aminotransferase activity in Table 13.
- the produced monatin and Trp were quantified using an ACQUITY UPLC system manufactured by Waters. The measurement conditions are shown below. After 15 minutes of reaction at 0.2 ml, the reaction was stopped, the reaction solution after the reaction was stopped, and about 0.2 ml of the supernatant was subjected to UPLC analysis.
- 2S, 4R-monatin can be fractionally quantified to 1.1 min
- 2S, 4S-monatin can be fractionated to 1.5 min
- Trp can be fractionated to 1.3 min.
- Example 30 pET-22-3976AT-His / E. 2S, 4R-monatin synthesis reaction using E. coli BL21 (DE3) pET-22-3976AT-His / E. E. coli BL21 (DE3) was inoculated into 3 ml of Overnight Express Instant Medium (Novagen) containing 100 mg / l of ampicillin and shaken at 37 ° C. for 16 hours using a test tube. After completion of the culture, the cells were collected from 1 ml of the obtained culture solution by centrifugation and suspended in 1 ml of BugBuster Master Mix (Novagen).
- Example 31 Rhizobium sp. Purification of AJ12469-derived aminotransferase Rhizobium sp. Purification of aminotransferase that produces 2S, 4R-monatin from the soluble fraction of AJ12469 was performed as follows. In the same manner as in Example 25, 2S, 4R-monatin synthesis reaction and 2S, 4R-monatin were quantified.
- Rhizobium sp. AJ12469 was applied and cultured at 30 ° C. for 2 days.
- Enzyme production medium yeast extract 10 g / l, tryptone 10 g / l, glucose 1 g / l, dipotassium hydrogen phosphate 3 g / l, potassium dihydrogen phosphate 1 g / l, magnesium sulfate heptahydrate
- One platinum loop was inoculated into 160 ml of 0.1 g / l, ammonium sulfate 5 g / l), and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C. for 16 hours.
- Bacterial cells were collected from about 1920 ml of the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), and sonicated at 4 ° C. for 30 minutes. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- Example 32 Rhizobium sp. Determination of N-terminal amino acid sequence of aminotransferase derived from AJ12469 When the purified enzyme solution obtained in Example 31 was subjected to N-terminal amino acid sequence analysis, the N-terminal amino acid sequence of AFLADILSRVKPSIATIVTQ (SEQ ID NO: 51) was obtained.
- the N-terminal amino acid sequence is Agrobacterium tumefaciens str. It showed high homology with C58-derived aspartate aminotransferase (AAK87940).
- Example 33 Rhizobium sp. Cloning of AJ12469-derived aminotransferase gene Rhizobium radiobacter AJ3976 was cultured in the same manner as in Example 31. Bacteria were collected from the obtained culture solution by centrifugation, and genomic DNA was extracted. A DNA fragment containing the aminotransferase gene was PCR amplified using the obtained genomic DNA as a template. Primers were obtained from Agrobacterium tumefaciens str.
- the primer Ag-u100-f (5'-ctgggtcagatagccggctttttgacc-3 ': SEQ ID NO: 45) and the primer Ag- designed from the 100 bp upstream and 100 bp downstream DNA sequences of the aminotransferase gene d100-r (5′-ccactttcatcatgctgctgtttctcg-3 ′: SEQ ID NO: 46) was used.
- PCR was performed using KOD-plus-ver. 2 (Toyobo) was used under the following conditions. 1 cycle 94 ° C, 2 min 25 cycles 98 ° C, 10 sec 55 ° C, 10 sec 68 °C, 60sec 1 cycle 68 ° C, 60 sec 4 °C
- Example 34 E.M. Rhizobium sp. AJ12469-derived aminotransferase expression (1) Rhizobium sp. Construction of AJ12469-derived aminotransferase expression plasmid Rhizobium sp. Using the genomic DNA of AJ12469 as a template, Rhizobium sp. A DNA fragment containing the AJ12469-derived aminotransferase gene was PCR amplified.
- Primers 12469AT-Nde-f (5′-ggaattccatATGGCCTCTCCTGCCCGACATTCCT-3 ′: SEQ ID NO: 54) and primers 12469-xho-r (5′-actccgctcgagGCGGCAATCGGCGCAAAACGCTGA-3 ′: SEQ ID NO: 55) were used. PCR was performed using KOD-plus-ver. 2 (Toyobo) was used under the following conditions. 1 cycle 94 ° C, 2 min 25 cycles 98 ° C, 10 sec 55 ° C, 10 sec 68 °C, 60sec 1 cycle 68 ° C, 60 sec 4 °C
- the obtained DNA fragment was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI.
- pET-22b Novagen
- the target plasmid was extracted from the ampicillin resistant strain, and this plasmid was named pET-22-12469AT-His.
- Rhizobium sp With His-tag added to the C-terminus.
- AJ12469-derived aminotransferase (12469AT-His) is expressed.
- E.E. 12469AT-His purification from E. coli expression strain The constructed expression plasmid pET-22-12469AT-His was obtained from E. coli.
- E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 160 ml of Overnight Express TB Medium (Novagen) containing ampicillin 100 mg / l and shaken at 37 ° C. for 16 hours using a 500 ml Sakaguchi flask.
- the cells were collected from about 1000 ml of the obtained culture solution by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, 20 mM Imidozole, and sonicated at 4 ° C. for 30 minutes. did.
- the cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- the obtained soluble fraction was mixed with His-tag protein purification column HisPrep FF 16/10 (Pharmacia (GE Healthcare Biosciences), CV, equilibrated with 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and 20 mM Imidazole.
- Example 35 Specific activity measurement results of AJ12469LAT for various keto acids (1) L-Asp / ⁇ -KG, L-Asp / PA, and L-Asp / ( ⁇ ) -MHOG activity measurement by colorimetric method AJ12469LAT Activity measurements on various substrates were performed. As the amino donor substrate for the transamination reaction, 100 mM L-Asp was used, and the specific activity against 10 mM of various keto acids was measured by a colorimetric method.
- L-Asp / ⁇ -KG activity L-Asp-Na-1aq 100 mM, ⁇ -KG-2Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH 2 U / ml, 25 ° C. . The activity is calculated from the decrease of 340 nm. MDH used Malic Dehydrogenase from porcine heart (Sigma). The L-Asp / ⁇ -KG activity is shown in the item “ ⁇ -KG” of aminotransferase activity in Table 15.
- L-Asp / PA activity L-Asp-Na-1aq 100 mM, PA-Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH (same as above) 2 U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm.
- the L-Asp / PA activity is shown in the item “PA” of aminotransferase activity in Table 15.
- L-Asp / ( ⁇ ) -MHOG activity L-Asp-Na-1aq 100 mM, ( ⁇ ) -MHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH (same as above) ) 2U / ml, LDH 10U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm.
- LDH D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used. LDH was added to remove PA mixed in a small amount in ( ⁇ ) -MHOG.
- the L-Asp / ( ⁇ ) -MHOG activity is shown in the item “ ⁇ MHOG” of aminotransferase activity in Table 15.
- L-Asp / 4R-IHOG, L-Asp / ( ⁇ ) -IHOG, and L-Asp / IPA activity measurement 2S, 4R-monatin producing activity from 4R-IHOG which is the target activity, ( ⁇ )- 2S, 4R-monatin and 2S, 4S-monatin producing activity from IHOG, and L-Trp side-life from IPA were measured, respectively.
- the amino donor substrate for the transamination reaction 100 mM L-Asp was used, transamination reaction was performed on 10 mM of various keto acids, the amount of amino acid produced was quantified by UPLC, and the specific activity was calculated.
- L-Asp / 4R-IHOG activity L-Asp-Na-1aq 100 mM, 4R-IHOG 10 mM (including a trace amount of 4S-IHOG), PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, 25 ° C. UPLC analysis was performed, and the produced 2S, 4R-monatin and 2S, 4S-monatin were quantified. As the reaction stop solution, a 200 mM Na citrate solution (pH 4.5) was used. The L-Asp / 4R-IHOG activity is shown in the item “4R-IHOG” of aminotransferase activity in Table 15.
- L-Asp / ( ⁇ ) -IHOG activity L-Asp-Na-1aq 100 mM, ( ⁇ ) -IHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, 25 ° C. UPLC analysis was performed, and the produced 2S, 4R-monatin and 2S, 4S-monatin were quantified. As the reaction stop solution, a 200 mM Na citrate solution (pH 4.5) was used. The L-Asp / ( ⁇ ) -IHOG activity is shown in the item “ ⁇ IHOG” of the aminotransferase activity in Table 13.
- L-Asp / IPA activity L-Asp-Na-1aq 100 mM, IPA 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM (pH adjusted to 8.0 with 1N NaOH after preparation of reaction solution), 25 ° C. .
- UPLC analysis was performed and the produced Trp was quantified.
- As the reaction stop solution a 200 mM Na citrate solution (pH 4.5) was used.
- the L-Asp / IPA activity is shown in the item “IPA” of aminotransferase activity in Table 15.
- the produced monatin and Trp were quantified using an ACQUITY UPLC system manufactured by Waters. The measurement conditions are shown below. After 15 minutes of reaction at 0.2 ml, the reaction was stopped, the reaction solution after the reaction was stopped, and about 0.2 ml of the supernatant was subjected to UPLC analysis.
- 2S, 4R-monatin can be fractionated and quantified to 1.1 min, 2S, 4S-monatin to 1.5 min, and Trp to 1.3 min.
- Example 36 pET-22-12469AT-His / E. 2S, 4R-monatin synthesis reaction using E. coli BL21 (DE3) pET-22-12469AT-His / E. E. coli BL21 (DE3) was inoculated into 3 ml of Overnight Express Instant Medium (Novagen) containing 100 mg / l of ampicillin and shaken at 37 ° C. for 16 hours using a test tube. After completion of the culture, the cells were collected by centrifugation from 1 ml of the obtained culture solution and suspended in 1 ml of BugBuster Master Mix (Novagen).
- Example 37 Purification of aminotransferase derived from Corynebacterium ammoniagenes AJ1444 The aminotransferase producing 2S, 4R-monatin was purified from the soluble fraction of Corynebacterium ammoniagenes AJ1444 as follows. In the same manner as in Example 25, 2S, 4R-monatin synthesis reaction and 2S, 4R-monatin were quantified.
- Corynebacterium ammoniagenes AJ1444 was apply
- Enzyme production medium yeast extract 10 g / l, tryptone 10 g / l, glucose 1 g / l, dipotassium hydrogen phosphate 3 g / l, potassium dihydrogen phosphate 1 g / l, magnesium sulfate heptahydrate
- One platinum loop was inoculated into 160 ml of 0.1 g / l, ammonium sulfate 5 g / l), and cultured with shaking in a 500 ml Sakaguchi flask at 30 ° C.
- the bacterial cells were collected from about 1760 ml of the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), added with glass beads, and disrupted with a multi-bead shocker (Yasui Kikai). did. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- Ammonium sulfate precipitation Ammonium sulfate was added to the soluble fraction so as to contain 90% (w / w) ammonium sulfate, and ammonium sulfate precipitation was obtained by centrifugation.
- Example 38 Determination of N-terminal amino acid sequence of aminotransferase derived from Corynebacterium ammoniagenes AJ1444
- the N-terminal amino acid sequence of MSXIAQXILDQ SEQ ID NO: 112
- the N-terminal amino acid sequence showed high homology with Corynebacterium striatum ATCC 6940-derived aspartate aminotransferase (ZP — 0395516) and Corynebacterium ammoniagenes DSM20306-derived aspartate aminotransferase (ZP — 06838515).
- Example 39 Cloning of Corynebacterium ammoniagenes AJ1444-derived aminotransferase gene
- Corynebacterium ammoniagenes AJ1444 was cultured in the same manner as in Example 37. Bacteria were collected from the obtained culture solution by centrifugation, and genomic DNA was extracted. A DNA fragment containing the aminotransferase gene was PCR amplified using the obtained genomic DNA as a template.
- Primers were designed from the DNA sequence of 50 bp downstream of the aminotransferase gene with reference to the genomic DNA sequence of Corynebacterium ammoniagenes DSM20306, primers Co-d50-r (5′-ctttctgtgaacagagtcgagagac-3 ′: SEQ ID NO: 56), and terneum Primer Co-800-f (5 ′) designed with reference to a highly homologous partial sequence of aspartate aminotransferase derived from STRATUM ATCC 6940 (ZP — 0395516) and Corynebacterium ammoniagenes DSM20306 aspartate aminotransferase (ZP — 06838515) gctatcgcacaattccaccgcacctt-3 ': using the SEQ ID NO: 57).
- PCR was performed using KOD-plus-ver. 2 (Toyobo) was used under the following conditions. 1 cycle 94 ° C, 2 min 25 cycles 98 ° C, 10 sec 55 ° C, 10 sec 68 °C, 60sec 1 cycle 68 ° C, 60 sec 4 °C
- the base sequence of the amplified DNA fragment of about 400 bp was determined, and based on the sequence, primer Co-890-r (5′-acactcgttaagcaagcgaaccaccag-3 ′: SEQ ID NO: 58) and primer Co-1060-r (5 ′ -Gaagagaagcgaatgtggtgctcg-3 ': SEQ ID NO: 59) was designed and PCR was performed using LA PCR in vitro Cloning Kit (Takara). PCR was performed using KOD-plus-ver. 2 (Toyobo) was used under the following conditions. 1 cycle 94 ° C, 2 min 25 cycles 98 ° C, 10 sec 55 ° C, 10 sec 68 °C, 60sec 1 cycle 68 ° C, 60 sec 4 °C
- HMPREF0281 — 0480 aspartate aminotransferase gene derived from Corynebacterium ammoniagenes DSM20306 was clarified (SEQ ID NOs: 60 and 61). The homology was 76% for the DNA sequence and 82% for the amino acid sequence. Since it also coincided with the N-terminal amino acid sequence obtained in Example 38, it was considered that an aminotransferase gene having 2S, 4R-monatin producing activity could be obtained.
- Example 40 E.I. Expression of Corynebacterium ammoniagenes AJ1444-derived aminotransferase in E. coli (1) Construction of Corynebacterium ammoniagenes AJ1444-derived aminotransferase expression plasmid Amplification of Corynebacterium ammoniagenes AJ1444 as a template DNA Primers 1444AT-Nde-f (5′-ggaattccatATGAGCCACATCGCTCAACCGCATCC-3 ′: SEQ ID NO: 62) and primer 1444-xho-r (5′-actccgctcgagGGACTTTGCGAAGATTTGGCGAATG-3 ′: SEQ ID NO: 63) were used.
- PCR was performed using KOD-plus-ver. 2 (Toyobo) was used under the following conditions. 1 cycle 94 ° C, 2 min 25 cycles 98 ° C, 10 sec 55 ° C, 10 sec 68 °C, 60sec 1 cycle 68 ° C, 60 sec 4 °C
- the obtained DNA fragment was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI.
- pET-22b Novagen
- the target plasmid was extracted from the ampicillin resistant strain, and this plasmid was named pET-22-1444AT-His.
- Corynebacterium amoniagenes AJ1444-derived aminotransferase (1444AT-His) with His-tag added to the C-terminus is expressed.
- E.E. 1444AT-His purification from E. coli expression strain The constructed expression plasmid pET-22-1444AT-His was obtained from E. coli.
- E. coli BL21 (DE3) was introduced, and the transformant was inoculated into 160 ml of Overnight Express TB Medium (Novagen) containing ampicillin 100 mg / l and shaken at 37 ° C. for 16 hours using a 500 ml Sakaguchi flask.
- the cells were collected from about 1000 ml of the obtained culture solution by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, 10 mM Imidozole, and ultrasonically disrupted at 4 ° C. for 30 minutes. did. The cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- the obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidozole 10 mM, and was adsorbed on the carrier.
- Example 41 Specific activity measurement results of AJ1444LAT for various keto acids (1) Colorimetric L-Asp / ⁇ -KG, L-Asp / PA, L-Asp / ( ⁇ ) -MHOG, L-Glu / Measurement of PA and L-Glu / ⁇ MHOG activity Activity of AJ1444LAT against various substrates was measured. As the amino donor substrate for transamination reaction, 100 mM L-Asp or L-Glu was used, and the specific activity against 10 mM of various keto acids was measured by a colorimetric method.
- L-Asp / ⁇ -KG activity L-Asp-Na-1aq 100 mM, ⁇ -KG-2Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH 2 U / ml, 25 ° C. . The activity is calculated from the decrease of 340 nm. MDH used Malic Dehydrogenase from porcine heart (Sigma). The L-Asp / ⁇ -KG activity is shown in the item “ ⁇ -KG” of aminotransferase activity in Table 17.
- L-Asp / PA activity L-Asp-Na-1aq 100 mM, PA-Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH (same as above) 2 U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm.
- the L-Asp / PA activity is shown in the item “PA” of aminotransferase activity in Table 17.
- L-Asp / ( ⁇ ) -MHOG activity L-Asp-Na-1aq 100 mM, ( ⁇ ) -MHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH (same as above) ) 2U / ml, LDH 10U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm. For LDH, D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used. LDH was added to remove PA mixed in a small amount in ( ⁇ ) -MHOG.
- L-Asp / ( ⁇ ) -MHOG activity is shown in the item “ ⁇ MHOG” of the aminotransferase activity in Table 17.
- L-Glu / PA activity L-Glu-Na 100 mM, PA 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NH 4 Cl 100 mM, NADH 0.25 mM, GDH 10 U / ml, 25 ° C. The activity is calculated from the decrease of 340 nm. GDH used L-Glutemic Dehydrogenase from bovine river (Sigma). The L-Glu / PA activity is shown in the item “PA” of aminotransferase activity in Table 17.
- L-Glu / ( ⁇ ) -MHOG activity L-Glu-Na 100 mM, ( ⁇ ) -MHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NH 4 Cl 100 mM, NADH 0.25 mM, GDH 10 U / Ml, 25 ° C. The activity is calculated from the decrease of 340 nm.
- the L-Glu / ( ⁇ ) -MHOG activity is shown in the item “ ⁇ MHOG” of the aminotransferase activity in Table 17.
- L-Asp / 4R-IHOG L-Asp / ( ⁇ ) -IHOG, L-Asp / IPA, L-Glu / 4R-IHOG, and L-Glu / IPA activity measurement 4R-IHOG which is the target activity 2S, 4R-monatin producing activity from 2 ⁇ , 2S, 4R-monatin and 2S, 4S-monatin producing activity from ( ⁇ ) -IHOG, and L-Trp side-life from IPA were measured.
- L-Asp or L-Glu As the amino donor substrate for the transamination reaction, 100 mM L-Asp or L-Glu was used, transamination reaction was performed on 10 mM of various keto acids, and the amount of amino acid produced was quantified by UPLC, and the specific activity was calculated.
- L-Asp / 4R-IHOG activity L-Asp-Na-1aq 100 mM, 4R-IHOG 10 mM (including a trace amount of 4S-IHOG), PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, 25 ° C. UPLC analysis was performed, and the produced 2S, 4R-monatin and 2S, 4S-monatin were quantified.
- L-Asp / 4R-IHOG activity is shown in the item “4R-IHOG” of aminotransferase activity in Table 17.
- L-Asp / ( ⁇ ) -IHOG activity L-Asp-Na-1aq 100 mM, ( ⁇ ) -IHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, 25 ° C.
- UPLC analysis was performed, and the produced 2S, 4R-monatin and 2S, 4S-monatin were quantified.
- reaction stop solution a 200 mM Na citrate solution (pH 4.5) was used.
- the L-Asp / ( ⁇ ) -IHOG activity is shown in the item “ ⁇ IHOG” of aminotransferase activity in Table 17.
- L-Asp / IPA activity L-Asp-Na-1aq 100 mM, IPA 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM (pH adjusted to 8.0 with 1N NaOH after preparation of reaction solution), 25 ° C. .
- UPLC analysis was performed and the produced Trp was quantified.
- As the reaction stop solution a 200 mM Na citrate solution (pH 4.5) was used.
- L-Asp / IPA activity is shown in the item “IPA” of aminotransferase activity in Table 17.
- L-Glu / 4R-IHOG activity L-Glu-Na 100 mM, 4R-IHOG 10 mM (including a trace amount of 4S-IHOG), PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, 25 ° C. UPLC analysis was performed, and the produced 2S, 4R-monatin and 2S, 4S-monatin were quantified. As the reaction stop solution, a 200 mM Na citrate solution (pH 4.5) was used.
- the L-Glu / 4R-IHOG activity is shown in the item “4R-IHOG” of the aminotransferase activity in Table 17.
- L-Glu / IPA activity L-Glu-Na 100 mM, IPA 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM (pH adjusted to 8.0 with 1N NaOH after preparation of the reaction solution), 25 ° C. UPLC analysis was performed and the produced Trp was quantified. As the reaction stop solution, a 200 mM Na citrate solution (pH 4.5) was used.
- the L-Glu / IPA activity is shown in the item “IPA” of aminotransferase activity in Table 17.
- Quantification of the produced monatin and Trp was carried out using an ACQUITY UPLC system manufactured by Waters. The measurement conditions are shown below. After 15 minutes of reaction at 0.2 ml, the reaction was stopped, the reaction solution after the reaction was stopped, and about 0.2 ml of the supernatant was subjected to UPLC analysis.
- 2S, 4R-monatin can be fractionated and quantified to 1.1 min, 2S, 4S-monatin to 1.5 min, and Trp to 1.3 min.
- Example 42 pET-22-1444AT-His / E. 2S, 4R-monatin synthesis reaction using E. coli BL21 (DE3) pET-22-1444AT-His / E. E. coli BL21 (DE3) was inoculated into 3 ml of Overnight Express Instant Medium (Novagen) containing 100 mg / l of ampicillin and shaken at 37 ° C. for 16 hours using a test tube. After completion of the culture, the cells were collected by centrifugation from 1 ml of the obtained culture solution and suspended in 1 ml of BugBuster Master Mix (Novagen).
- Example 43 One-pot synthesis reaction of 2S, 4R-monatin from L-Trp 20 mM (AJ3976LAT, AJ12469LAT, AJ1444LAT) Using purified 3976AT-His, 12469AT-His, and 1444AT-His, the reaction was performed for 12 hours under the following conditions. The reaction was performed in 1 ml using a test tube. Sampling was performed appropriately, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the filtrate was analyzed. Analysis was performed by HPLC and capillary electrophoresis.
- Reaction conditions L-Trp 20 mM, PA-Na 40 mM, L-Asp-Na-1aq 160 mM, MgCl 2 1 mM, PLP 50 ⁇ M, Tris-HCl 100 mM, KPB 20 mM, pH 7.0, Ps_aad broth 20%, SpAld purified enzyme 30 U / Ml, OAA DCase commercial enzyme 10 U / ml, LAT purified enzyme 2 U / ml (vs 10 mM 4R-IHOG), SOD commercial enzyme 200 U / ml, 25 ° C., 120 rpm.
- Ps_aad broth prepared according to the method described in Example 17.
- SpAld purified enzyme prepared according to the method described in Example 19.
- AJ3976LAT, AJ12469LAT, AJ1444LAT Prepared according to the methods described in Examples 28, 34, 40.
- OAA DCase Oxaloacetate Decaboxylase from Pseudomonas sp. (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- SOD Superoxide Dissimilar from bovine river (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- Example 44 One-pot synthesis reaction of 2S, 4R-monatin from 50 mM Trp (AJ3976, 80 ml scale) Using purified 3976AT-His, the reaction was carried out for 12 hours under the following conditions. The reaction was carried out at 80 ml using a 250 ml mini jar. Sampling was performed appropriately, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the filtrate was analyzed. Analysis was performed by HPLC and capillary electrophoresis.
- Ps_aad broth prepared according to the method described in Example 17.
- SpAld purified enzyme prepared according to the method described in Example 19.
- AJ3976LAT Prepared according to the method described in Example 28.
- OAA DCase Oxaloacetate Decaboxylase from Pseudomonas sp. (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- SOD Superoxide Dissimilar from bovine river (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- Example 45 E.I. Expression of in silico selected aminotransferase in E. coli (1) Construction of in silico selected aminotransferase expression plasmid NdeI recognition sequence at 5 ′ end of gene sequence of various aminotransferases selected from in silico and XhoI recognition sequence at 3 ′ end The DNA sequence provided with the above was subjected to GenScript Optimum Codon Optimization Analysis. Synthetic DNA with optimized gene expression efficiency in E. coli was obtained. The types of aminotransferase are as follows.
- Aspartate Aminotransferase (Ere, ACR74350) (SEQ ID NO: 86, 87), Bacillus pumilus SAFR-032 derived Aspartate Aminotransferase (Bpu, ABV62783) (SEQ ID NO: 88, 89), Bacillus cell Lostilyticus DSM 2522-derived Putative transcriptional regulator, GntR family (Bce, ADU 30616) (SEQ ID NO: 90, 91), Bacillus specials (strain YM-2), Aspartate 93 1021-derived Aspartate Aminotransferase aatB (SmeB, CAC47870) (SEQ ID NOs: 94, 95), Methanotherbacter thermotrophicus str.
- Aspartate Aminotransferase (Ere, ACR74350) (SEQ ID NO: 86, 87), Bacillus pumilus SAFR-032 derived Aspartate Aminotransferase (B
- Delta H-derived Branched-chain amino-acid aminotransferase (SEQ ID NO: 96, 97), Lactobacillus acidofilus-derived Aspartate aminoflase (Lba, AAV43507) (Lba, AAV43507) , CAC46904) (SEQ ID NO: 100, 101), Pyrococcus horikoshi OT3-derived hypothetical serine aminotransferase (PhoS, BAA30413) (SEQ ID NO: 102, 103), Thermo anaerobacter tengcongensis MB4 from PLP-dependent aminotransferases (Tte, AAM24436) (SEQ ID NO: 104,105), Clostridium cellulolyticum H10 derived Putative transcriptional regulator, GntR family (Cce, ACL75101) (SEQ ID NO: 106,107), Rhodococcus erythropolis PR4 from Aspartate aminotransferase As
- the synthetic DNA was treated with restriction enzymes with NdeI and XhoI, respectively, and similarly ligated with pET-22b (Novagen) treated with NdeI and XhoI.
- pET-22b Novagen
- NdeI and XhoI With this ligation solution E. coli JM109 was transformed, and the target plasmid was extracted from ampicillin resistant strains.
- These plasmids were named pET-22-AT-His.
- These plasmids express various aminotransferases (AT-His) with His-tag added to the C-terminus.
- the shaking temperature is 25 ° C for Lba, Dge, Pla, Tth, Tma2, Sce, Ere, Bpu, Bce, Bsp, SmeA, PhoS, Rer, Sde for 30 ° C, Cgl, TtHB, PhoH, PhoA, SmeB, Tte, Cce was performed at 37 ° C., and Tma1, Mja, and Mth were performed at 42 ° C.
- bacterial cells were collected from the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic crushing.
- the cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
- the obtained soluble fraction was subjected to His-tag protein purification column His TALON superflow 5 ml Cartridge (Clontech) equilibrated with Tris-HCl (pH 7.6) 20 mM, NaCl 300 mM, and Imidazole 10 mM, and was adsorbed on the carrier.
- Example 46 One-pot synthesis reaction of 2S, 4R-monatin from 20 mM L-Trp Reaction was carried out for 15 hours under the following conditions using various purified AT-His. The reaction was performed in 1 ml using a test tube. After completion of the reaction, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the filtrate was analyzed. Analysis was performed by HPLC and capillary electrophoresis.
- Reaction conditions L-Trp 20 mM, PA-Na 40 mM, L-Asp-Na-1aq 160 mM, MgCl 2 1 mM, PLP 50 ⁇ M, Tris-HCl 100 mM, KPB 20 mM, pH 7.0, Ps_aad broth 20%, SpAld purified enzyme 30 U / Ml, OAA DCase commercial enzyme 10 U / ml, LAT purified enzyme 1 mg / ml, SOD commercial enzyme 200 U / ml, 25 ° C., 120 rpm.
- Ps_aad broth prepared according to the method described in Example 17.
- SpAld purified enzyme prepared according to the method described in Example 19.
- Various LATs prepared according to the method described in Example 45.
- OAA DCase Oxaloacetate Decaboxylase from Pseudomonas sp. (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- SOD Superoxide Dissimilar from bovine river (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- Example 47 One-pot synthesis reaction of 2S, 4R-monatin from L-Trp 20 mM (Tth, Bpu, SmeA, Sde) Using various purified AT-His, the reaction was carried out for 15 hours under the following conditions. The reaction was performed in 1 ml using a test tube. After completion of the reaction, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the filtrate was analyzed. Analysis was performed by HPLC and capillary electrophoresis.
- Reaction conditions L-Trp 20 mM, PA-Na 40 mM, L-Asp-Na-1aq 160 mM, MgCl 2 1 mM, PLP 50 ⁇ M, Tris-HCl 100 mM, KPB 20 mM, pH 7.0, Ps_aad broth 20%, SpAld purified enzyme 30 U / Ml, OAA DCase commercial enzyme 10 U / ml, LAT purified enzyme 3 mg / ml (Tth is 12 mg / ml, Bpu is 1 mg / ml), SOD commercial enzyme 200 U / ml, 25 ° C., 120 rpm.
- Ps_aad broth prepared according to the method described in Example 17.
- SpAld purified enzyme prepared according to the method described in Example 19.
- Various LATs prepared according to the method described in Example 45.
- OAA DCase Oxaloacetate Decaboxylase from Pseudomonas sp. (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- SOD Superoxide Dissimilar from bovine river (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- Example 48 One-pot synthesis reaction of 2S, 4R-monatin from L-Trp 100 mM (Tth, Bpu, SmeA, Sde) Using AT-His purified from Tth, Bpu, SmeA, and Sde, the reaction was performed for 18 hours under the following conditions. The reaction was performed in 1 ml using a test tube. After completion of the reaction, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa (Millipore), and the filtrate was analyzed. Analysis was performed by HPLC and capillary electrophoresis.
- Reaction conditions L-Trp 100 mM, PA-Na 50 mM, L-Asp-Na 300 mM, MgCl 2 1 mM, PLP 50 ⁇ M, Tris-HCl 100 mM, KPB 20 mM, pH 7.0, Ps_aad broth 40%, SpAld purified enzyme 60 U / ml , OAA DCase commercial enzyme 10 U / ml, LAT purified enzyme 3 mg / ml (Tth is 12 mg / ml), SOD commercial enzyme 200 U / ml, 25 ° C., 150 rpm.
- Ps_aad broth prepared according to the method described in Example 17.
- SpAld purified enzyme prepared according to the method described in Example 19.
- Various LATs prepared according to the method described in Example 45.
- OAA DCase Oxaloacetate Decaboxylase from Pseudomonas sp. (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- SOD Superoxide Dissimilar from bovine river (Sigma) was used.
- the enzyme amount (U) used was the value described by the manufacturer.
- the microorganism specified by the accession number described in this specification can be obtained from a predetermined accession organization by referring to the accession number.
- the microorganisms listed in Table 22 are listed below at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, Japan), which is an international depositary organization under the Budapest Treaty. The following deposit number is attached.
- these microorganisms have conventionally been given different names, but as a result of re-identification, they are currently classified as follows.
- microorganisms listed in Table 23 have conventionally been given different names, but as a result of re-identification, they are currently classified as follows.
- strain Stenotrophomonas sp. AJ13127 is identical to a known strain identified by accession number FERM-BP5568.
- the method of the present invention is useful for producing monatin that can be used as a sweetener.
- SEQ ID NO: 1 Nucleotide sequence of an aminotransferase gene derived from Bacillus altitudini
- SEQ ID NO: 2 Amino acid sequence of an aminotransferase derived from Bacillus alterdinis
- SEQ ID NO: 3 An aminotransferase gene derived from Bacillus altitudinis (base numbers 231 to 1538) and upstream thereof
- the nucleotide sequence of the downstream region SEQ ID NO: 4: amino acid sequence of an aminotransferase fragment derived from Bacillus altitudini
- SEQ ID NO: 5 the amino acid sequence of an aminotransferase fragment derived from Bacillus altitudini
- SEQ ID NO: 6 a DNA flag containing an aminotransferase gene derived from Bacillus altitudinis Forward primer for amplifying the cement (Bp-u200-f)
- SEQ ID NO: 7 Reverse primer (Bp-d200-r) for amplifying a DNA
- Amino acid sequence of aminotransferase fragment derived from SEQ ID NO: 52 Rhizobium sp.
- Amino acid sequence of aminotransferase derived from SEQ ID NO: 54 Rhizobium sp.
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Abstract
Description
しかしながら、アルドラーゼ工程(第二工程)は平衡反応ゆえ、必ずしも満足いく収率が得られる反応とは言えなかった。
〔1〕L-アミノ酸の存在下において、4R-IHOGをL-アミノ酸アミノトランスフェラーゼに接触させて、2S,4R-モナティンを生成することを含む、2S,4R-モナティンまたはその塩の製造方法。
〔2〕L-アミノ酸アミノトランスフェラーゼの作用によりL-アミノ酸から生成したケト酸を、デカルボキシラーゼに接触させて分解することをさらに含む、〔1〕の製造方法。
〔3〕L-アミノ酸がL-アスパラギン酸である、〔1〕の製造方法。
〔4〕L-アミノ酸アミノトランスフェラーゼの作用によりL-アスパラギン酸から生成したオキサロ酢酸を、オキサロ酢酸デカルボキシラーゼに接触させて、ピルビン酸を不可逆的に生成することをさらに含む、〔3〕の製造方法。
〔5〕L-アミノ酸アミノトランスフェラーゼが、アルスロバクター属、バチルス属、カンジダ属、コリネバクテリウム属、ロデロミセス属、マイクロコッカス属、マイクロバクテリウム属、ノカルディア属、シュードモナス属、リゾビウム属、ステノトロホモナス属、ディエジア属、オクロバクトラム属、ブレブンディモナス属、バークホルデリア属、カーニモナス属、ヤロウィア属、クロストリジウム属、デイノコッカス属、ユーバクテリウム属、ラクトバチルス属、メタノサーモバクター属、ホルミジウム属、ピロコッカス属、ロドコッカス属、サッカロマイセス属、サッカロファガス属、シノリゾビウム属、サーモアナエロバクター属、サーモトガ属、またはサーマス属に属する微生物に由来する、〔1〕の製造方法。
〔6〕L-アミノ酸アミノトランスフェラーゼが、アルスロバクター・エスピー、バチルス・アルティトゥディニス、バチルス・セルロシリティカス、バチルス・プミルス、バチルス・エスピー、カンジダ・ノルベゲンシス、カンジダ・インコンスピクア、コリネバクテリウム・アンモニアゲネス、コリネバクテリウム・グルタミカム、ロデロミセス・エロンギスポルス、マイクロコッカス・ルテウス、マイクロバクテリウム・エスピー、ノカルディア・グロベルラ、シュードモナス・クロロラフィス、シュードモナス・シトロノクロリス、シュードモナス・フラギ、シュードモナス・プチダ、シュードモナス・シンキサンタ、シュードモナス・タエトロレンス、シュードモナス・エスピー、リゾビウム・ラディオバクター、リゾビウム・エスピー、ステノトロホモナス・エスピー、ディエジア・マリス、オクロバクトラム・シュードグリグノネンス、ブレブンディモナス・ディミヌタ、バークホルデリア・エスピー、カーニモナス・エスピー、ヤロウィア・リポリティカ、クロストリジウム・セルロリティカム、デイノコッカス・ゲオサーマリス、ユーバクテリウム・レクタル、ラクトバチルス・アシドフィルス、メタノサーモバクター・サーマウトトロフィカス、ホルミジウム・ラピデウム、ピロコッカス・ホリコシイ、ロドコッカス・エリスロポリス、サッカロマイセス・セレビジアエ、サッカロファガス・デグラダンス、シノリゾビウム・メリロティ、サーモアナエロバクター・テングコンゲネシス、サーモトガ・マリチマ、またはサーマス・サーモフィラスに属する微生物に由来する、〔5〕の製造方法。
〔7〕L-アミノ酸アミノトランスフェラーゼが、配列番号2、配列番号48、配列番号53、配列番号61、配列番号65、配列番号67、配列番号69、配列番号73、配列番号75、配列番号77、配列番号83、配列番号85、配列番号87、配列番号89、配列番号91、配列番号93、配列番号95、配列番号97、配列番号99、配列番号101、配列番号103、配列番号105、配列番号107、配列番号109、または配列番号111により表されるアミノ酸配列に対して90%以上の同一性を示すアミノ酸配列からなる、〔1〕の製造方法。
〔8〕L-アミノ酸アミノトランスフェラーゼが、配列番号2により表されるアミノ酸配列において39位、109位、128位、150位、258位、287位、288位、289位、303位、358位、及び431位のアミノ酸残基から選ばれる1個以上のアミノ酸残基の変異を含む、〔7〕の製造方法。
〔9〕1個以上のアミノ酸残基の変異が、以下からなる群より選ばれる1個以上のアミノ酸残基の置換である、〔8〕の製造方法:
1)39位のリジンのアルギニンへの置換;
2)258位のセリンのグリシンへの置換;
3)287位のグルタミンのグルタミン酸への置換;
4)288位のスレオニンのグリシンへの置換;
5)289位のイソロイシンのアラニンへの置換;
6)109位のアスパラギン酸のグリシンへの置換;
7)150位のヒスチジンのチロシンへの置換;
8)303位のフェニルアラニンのロイシンへの置換;
9)358位のアスパラギン酸のチロシンへの置換;
10)431位のセリンのスレオニンへの置換;及び
11)128位のグルタミン酸のグリシンへの置換。
〔10〕L-アミノ酸アミノトランスフェラーゼを発現する形質転換体を用いて、4R-IHOGをL-アミノ酸アミノトランスフェラーゼに接触させる、〔1〕の製造方法。
〔11〕インドール-3-ピルビン酸およびピルビン酸を縮合して、4R-IHOGを生成することをさらに含む、〔1〕の製造方法。
〔12〕インドール-3-ピルビン酸およびピルビン酸をアルドラーゼに接触させることにより、インドール-3-ピルビン酸およびピルビン酸を縮合する、〔11〕の製造方法。
〔13〕4R-IHOGの生成に用いられるピルビン酸の少なくとも一部が、オキサロ酢酸デカルボキシラーゼの作用によりオキサロ酢酸から生成したピルビン酸に由来する、〔11〕の製造方法。
〔14〕トリプトファンを脱アミノ化して、インドール-3-ピルビン酸を生成することをさらに含む、〔11〕の製造方法。
〔15〕トリプトファンを脱アミノ化酵素に接触させることにより、トリプトファンを脱アミノ化する、〔14〕の製造方法。
〔16〕2S,4R-モナティンまたはその塩の製造が1つの反応槽中で行われる、〔11〕または〔14〕の製造方法。
〔17〕以下(I)および(II)を含む、2R,4R-モナティンまたはその塩の製造方法:
(I)〔1〕の方法により、2S,4R-モナティンを生成すること;および
(II)2S,4R-モナティンを異性化させて、2R,4R-モナティンを生成すること。
〔18〕芳香族アルデヒドの存在下において、2S,4R-モナティンを異性化する、〔17〕の製造方法。
〔19〕塩がナトリウム塩またはカリウム塩である、〔17〕の製造方法。
〔20〕下記(A)~(D)からなる群より選ばれるタンパク質である、L-アミノ酸アミノトランスフェラーゼ:
(A)配列番号2、配列番号48、配列番号53、または配列番号61により表されるアミノ酸配列からなるタンパク質;
(B)配列番号2、配列番号48、配列番号53、または配列番号61により表されるアミノ酸配列を含むタンパク質;
(C)配列番号2、配列番号48、配列番号53、または配列番号61により表されるアミノ酸配列に対して90%以上の同一性を示すアミノ酸配列からなり、かつL-アミノ酸アミノトランスフェラーゼ活性を有する、タンパク質;ならびに
(D)配列番号2、配列番号48、配列番号53、または配列番号61により表されるアミノ酸配列において、アミノ酸残基の欠失、置換、付加および挿入からなる群より選ばれる、1または数個のアミノ酸残基の変異を含むアミノ酸配列からなり、かつL-アミノ酸アミノトランスフェラーゼ活性を有する、タンパク質。
〔21〕L-アミノ酸アミノトランスフェラーゼが、配列番号2により表されるアミノ酸配列において39位、109位、128位、150位、258位、287位、288位、289位、303位、358位、及び431位のアミノ酸残基から選ばれる1個以上のアミノ酸残基の変異を含む、〔20〕のL-アミノ酸アミノトランスフェラーゼ。
〔22〕1個以上のアミノ酸残基の変異が、以下からなる群より選ばれる1個以上のアミノ酸残基の置換である、〔21〕のL-アミノ酸アミノトランスフェラーゼ:
1)39位のリジンのアルギニンへの置換;
2)258位のセリンのグリシンへの置換;
3)287位のグルタミンのグルタミン酸への置換;
4)288位のスレオニンのグリシンへの置換;
5)289位のイソロイシンのアラニンへの置換;
6)109位のアスパラギン酸のグリシンへの置換;
7)150位のヒスチジンのチロシンへの置換;
8)303位のフェニルアラニンのロイシンへの置換;
9)358位のアスパラギン酸のチロシンへの置換;
10)431位のセリンのスレオニンへの置換;及び
11)128位のグルタミン酸のグリシンへの置換。
〔23〕下記(a)~(e)からなる群より選ばれる、ポリヌクレオチド:
(a)配列番号1、配列番号47、配列番号52、または配列番号60により表されるヌクレオチド配列からなるポリヌクレオチド;
(b)配列番号1、配列番号47、配列番号52、または配列番号60により表されるヌクレオチド配列を含むポリヌクレオチド;
(c)配列番号1、配列番号47、配列番号52、または配列番号60により表されるアミノ酸配列に対して90%以上の同一性を示すヌクレオチド配列からなり、かつL-アミノ酸アミノトランスフェラーゼ活性を有するタンパク質をコードする、ポリヌクレオチド;
(d)配列番号1、配列番号47、配列番号52、または配列番号60により表されるヌクレオチド配列に対して相補的なヌクレオチド配列からなるポリヌクレオチドとストリンジェント条件下でハイブリダイズし、かつL-アミノ酸アミノトランスフェラーゼ活性を有するタンパク質をコードする、ポリヌクレオチド;ならびに
(e)〔20〕のL-アミノ酸アミノトランスフェラーゼをコードするポリヌクレオチド。
〔24〕〔23〕のポリヌクレオチドを含む発現ベクター。
〔25〕〔24〕の発現ベクターが導入された形質転換体。
〔26〕〔25〕の形質転換体を培地中で培養して、L-アミノ酸アミノトランスフェラーゼを得ることを含む、L-アミノ酸アミノトランスフェラーゼの製造方法。
〔27〕L-アミノ酸の存在下において、4R-IHOGを〔20〕のL-アミノ酸アミノトランスフェラーゼに接触させて、2S,4R-モナティンを生成することを含む、2S,4R-モナティンまたはその塩の製造方法。
〔28〕以下(I’)および(II’)を含む、2R,4R-モナティンまたはその塩の製造方法:
(I’)〔27〕の方法により、2S,4R-モナティンを生成すること;および
(II’)2S,4R-モナティンを異性化させて、2R,4R-モナティンを生成すること。
〔29〕芳香族アルデヒドの存在下において、2S,4R-モナティンを異性化する、〔28〕の製造方法。
〔30〕塩がナトリウム塩またはカリウム塩である、〔28〕の製造方法。
本発明は、2S,4R-モナティンまたはその塩の製造方法(1)を提供する。本発明の製造方法は、(1-1)4R-IHOGから2S,4R-モナティンを製造する方法、(1-2)IPAおよびピルビン酸から2S,4R-モナティンを製造する方法、(1-3)トリプトファンから2S,4R-モナティンを製造する方法に分類できる。(1-1)、(1-2)、(1-3)の方法は、L-アミノ酸の存在下において、4R-IHOGをL-アミノ酸アミノトランスフェラーゼに接触させて、2S,4R-モナティンを生成することを含むという点で共通する。
本方法は、L-アミノ酸の存在下において、4R-IHOGをL-アミノ酸アミノトランスフェラーゼに接触させて、2S,4R-モナティンを生成すること(反応1)を含む。L-アミノ酸の存在下において、4R-IHOGをL-アミノ酸アミノトランスフェラーゼに接触させることにより、L-アミノ酸のアミノ基が4R-IHOGに転移して、2S,4R-モナティンを生成し得る。
1)39位のリジンのアルギニンへの置換;
2)258位のセリンのグリシンへの置換;
3)287位のグルタミンのグルタミン酸への置換;
4)288位のスレオニンのグリシンへの置換;
5)289位のイソロイシンのアラニンへの置換;
6)109位のアスパラギン酸のグリシンへの置換;
7)150位のヒスチジンのチロシンへの置換;
8)303位のフェニルアラニンのロイシンへの置換;
9)358位のアスパラギン酸のチロシンへの置換;
10)431位のセリンのスレオニンへの置換;及び
11)128位のグルタミン酸のグリシンへの置換
a)T288G
b)S258G/I289A
c)K39R/T288G
d)Q287E/T288G
e)K39R/D109R/T288G/S431T
f)K39R/D109R/T288G/F303L
g)D109R/Q287E/T288G/F303L
h)D109R/S258G/I289A/F303L
i)D109R/Q287E/T288G/S431T
j)D109R/S258G/I289A/S431T
k)K39R/D109R/E128G/T288G/F303L
l)K39R/D109G/E128G/T288G/F303L
m)D109R/E128G/Q287E/T288G/F303L
n)D109R/E128G/S258G/I289A/S431T
o)D109G/E128G/Q287E/T288G/F303L
p)D109G/E128G/S258G/I289A/F303L
q)K39R/D109G/H150Y/T288G/F303L/D358Y/S431T
r)K39R/D109G/E128G/H150Y/T288G/F303L/D358Y
s)D109G/H150Y/Q287E/T288G/F303L/D358Y/S431T
t)D109G/H150Y/S258G/I289A/F303L/D358Y/S431T
u)D109G/E128G/H150Y/Q287E/T288G/F303L/D358Y
v)D109G/E128G/H150Y/S258G/I289A/F303L/D358Y
・L-アミノ酸アミノトランスフェラーゼ(抽出酵素)およびデカルボキシラーゼ(抽出酵素)
・L-アミノ酸アミノトランスフェラーゼの産生菌およびデカルボキシラーゼ(抽出酵素)
・L-アミノ酸アミノトランスフェラーゼ(抽出酵素)およびデカルボキシラーゼの産生菌
・L-アミノ酸アミノトランスフェラーゼの産生菌およびデカルボキシラーゼの産生菌
・L-アミノ酸アミノトランスフェラーゼおよびデカルボキシラーゼの産生菌
本発明の製造方法は、4R-IHOGを調製するために、IPAおよびピルビン酸を縮合させて、4R-IHOGを生成することをさらに含んでいてもよい。IPAおよびピルビン酸の縮合は、有機化学的手法、およびアルドラーゼを用いる酵素法により行うことができる。有機化学的手法によりIPAおよびピルビン酸を縮合させて4R-IHOGを生成する方法は、例えば、国際公開第2003/059865号、米国特許出願公開第2008/0207920号明細書に開示されている。アルドラーゼを用いる酵素法によりIPAおよびピルビン酸を縮合させて4R-IHOGを生成する方法は、例えば、国際公開第2003/056026号、特開2006-204285、米国特許出願公開第2005/0244939号明細書、国際公開第2007/103989号に開示されている。したがって、本発明では、IPAおよびピルビン酸から4R-IHOGを調製するために、これらの方法を用いることができる。
本発明の製造方法は、IPAを調製するために、トリプトファン(Trp)を脱アミノ化することをさらに含んでいてもよい。Trpとしては、L-Trp、D-Trp、ならびにL-TrpおよびD-Trpの混合物が挙げられる。Trpの脱アミノ化は、有機化学的手法、および脱アミノ化酵素を用いる酵素法により行うことができる。
アミノ酸デアミナーゼまたはその産生菌を用いてTrpからIPAを生成する方法としては、例えば、国際公開第2009/028338号に開示される方法が挙げられる。アミノ酸デアミナーゼ触媒する反応の一般式としては、以下が挙げられる<式:アミノ酸+H2O→2-オキソ酸+NH3>。
また、アミノトランスフェラーゼまたはその産生菌を用いてTrpからIPAを生成する方法としては、例えば、東ドイツ特許DD 297190、特開昭59-95894号公報、国際公開第2003/091396号、米国特許出願公開2005/0282260号明細書に開示される方法が挙げられる。
L-アミノ酸オキシダーゼまたはその産生菌を用いてTrpからIPAを生成する方法としては、例えば、米国特許第5,002,963号公報、John A.Duerreら(Journal of Bacteriology 1975,vol121,No.2,p656-663)、特開昭57-146573号公報、国際公開第2003/056026号、国際公開第2009/028338号に開示される方法が挙げられる。アミノ酸オキシダーゼの触媒する反応の一般式としては、以下が挙げられる<式:アミノ酸+O2+H2O→2-オキソ酸+H2O2+NH3>。この際、副生する過酸化水素による化合物の分解を抑制する目的で、カタラーゼなどの過酸化水素分解酵素を反応液に添加してもよい。
デアミナーゼ活性:10mM L-Phe、100mM NH4Cl、100mM Tris-HCl(pH8.0)、0.25mM NADH、フェニルアラニンデヒドロゲナーゼ(ユニチカ社製、Thermoactinomyces intermedius由来)、25℃。340nmの減少から活性を算出。
L-アミノ酸アミノトランスフェラーゼ活性(L-Asp/α-KG活性):100mM L-Asp-Na-1aq、10mM α-KG-2Na、50μM PLP、100mMTris-HCl(pH8.0)、0.25mM NADH、MDH 2U/ml、25℃。340nmの減少から活性を算出。MDHはMalic Dehydrogenase from porcine heart(Sigma)を用いた。
アルドラーゼ活性:2mM 4-phenyl-4-hydroxy-2-oxo glutarate(PHOG)、100mM Tris-HCl(pH7.0)、1mM MgCl2、0.25mM NADH、10U/ml lactate dehydrogenase(オリエンタル酵母社製、Leuconostoc mesenteroides由来),25℃。340nmの減少から活性を算出。
オキザロ酢酸デカルボキシラーゼ活性:1mM オキザロ酢酸、100mM Tris-HCl(pH8.0)、0.25mM NADH、10U/ml lactate dehydrogenase(オリエンタル酵母社製、Leuconostoc mesenteroides由来),25℃。340nmの減少から活性を算出。
このようにして決定された酵素活性に基づくと、反応液中に添加される酵素量は、以下のとおりであってもよい。反応液中に添加されるデアミナーゼの量は、例えば0.1~20U/ml、好ましくは0.5~2 U/mlである。反応液中に添加されるアルドラーゼの量は、例えば1~1000U/ml,好ましくは10~100U/mlである。反応液中に添加されるL-アミノ酸アミノトランスフェラーゼの量は、例えば1~1000U/ml、好ましくは10~100U/mlである。反応液中に添加されるオキザロ酢酸デカルボキシラーゼの量は、例えば0.01U/ml以上、好ましくは0.1U/ml以上である。各基質は、バッチ法またはフィード法により反応系に添加されてもよい。酵素、酵素発現菌体、酵素発現菌体処理物、培養ブロス、または培養ブロス処理物もまた、バッチ法またはフィード法により反応系に添加されてもよい。反応時間は、例えば2~100時間、好ましくは4~50時間、より好ましくは8~25時間である。反応液は、適切な条件(例、温度、pH、時間)下で殺菌されてもよい。
本発明は、2R,4R-モナティンまたはその塩の製造方法(2)を提供する。本発明の製造方法は、本発明の製造方法(1)により2S,4R-モナティンまたはその塩を製造すること、ならびに2S,4R-モナティンまたはその塩を異性化して、2R,4R-モナティンまたはその塩を生成することを含む。
実施例1~7では、HPLC分析を実施した場合、当該実施例に示す条件にてHPLC分析を実施した。
一方、実施例8~15では、以下に示す条件にてHPLC分析を実施した。
検出器:紫外吸光光度計(測定波長:210nm)
カラム温度:40℃
カラム :CAPCELLPAK C18 Type MGII,内径3mm,
長さ25cm,粒径5μm、資生堂(株)
移動相 :A液 20mM燐酸二水素カリウム水溶液:アセトニトリル=95:5
B液 20mM燐酸二水素カリウム水溶液:アセトニトリル=60:40
グラジエントプログラム:以下の表1を参照
注入量 :20μL
分析時間 :60分
CM2G寒天培地(酵母エキス 10g/l、ポリペプトン 10g/l、グルコース 5g/l、塩化ナトリウム 5g/l、寒天 15g/l、pH7.0)にBacillus altitudinis AJ1616を塗布し、30℃で2日間培養した。
得られた菌体を酵素生産培地(酵母エキス 10g/l、ポリペプトン 10g/l、グルコース 1g/l、リン酸水素二カリウム 3g/l、リン酸二水素カリウム 1g/l、硫酸マグネシウム七水和物 0.1g/l、硫酸アンモニウム 5g/l)3mlに一白金耳接種し、試験管で30℃にて16時間振とう培養した。培養液2mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mMにて洗浄、懸濁し、菌体懸濁液を1ml調製した。
この菌体懸濁液1mlにガラスビーズ(0.1mm)を1g添加し、マルチビーズショッカー(安井器械)を用いて菌体を破砕した。得られた破砕液を遠心分離し、上清を菌体抽出液とした。
2S,4R-モナティン合成反応液(4R-IHOG 9.5mM、4S-IHOG 0.5mM、L-Asp 100mM、PLP 50μM、Tris-HCl 100mM、pH8.0)0.1mlに、Bacillus altitudinis AJ1616菌体抽出液が0.05ml含まれるよう調製し、30℃で20時間反応させた。反応終了後、生成した2S,4R-モナティンを定量したところ、0.21mMであった。
移動相:20mM KH2PO4/アセトニトリル = 100/5
流速:0.15ml/min
カラム温度:40℃
検出:UV 210nm
カラム:ACQUITY UPLC BEH C18、2.1×50mm、1.7μm(ウォーターズ)
Bacillus altitudinis AJ1616の可溶性画分から、2S,4R-モナティンを生成するアミノトランスフェラーゼの精製を以下の通り行った。実施例1と同様にして、2S,4R-モナティン合成反応および2S,4R-モナティンの定量を行った。
CM2G寒天培地(酵母エキス 10g/l、ポリペプトン 10g/l、グルコース 5g/l、塩化ナトリウム 5g/l、寒天 15g/l、pH7.0)にBacillus altitudinis AJ1616を塗布し、30℃で2日間培養した。
得られた菌体をTB(Terrific Broth)培地160mlに一白金耳接種し、500ml容坂口フラスコで30℃にて16時間振とう培養した。得られた培養液約2000mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 100mMにて洗浄、懸濁し、4℃で30分間超音波破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
上記の可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 100mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 26/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=53ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 100mMで用いて洗い流した後、NaCl濃度を100mMから500mMまで直線的に変化させて、8ml/minの流速で吸着したタンパク質の溶出を行った。各溶出画分について2S,4R-モナティン生成活性を検出したところ、約200mM NaCl相当の画分に2S,4R-モナティン生成活性が認められた。
2S,4R-モナティン生成活性が検出された画分を集めて、硫酸アンモニウム 1.4M、Tris-HCl 20mM(pH7.6)となるよう、硫酸アンモニウム、Tris-HCl(pH7.6)を添加した。この溶液を硫酸アンモニウム 1.4M、Tris-HCl 20mM(pH7.6)で平衡化した疎水性クロマトグラフィーカラムHiLoad 16/10 Phenyl Sepharose HP(GEヘルスケアバイオサイエンス社製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかった非吸着タンパク質を硫酸アンモニウム1.4M、Tris-HCl(pH7.6) 20mMを用いて洗い流した後、硫酸アンモニウム濃度を1.4Mから0Mまで直線的に変化させて、3ml/minの流速で2S,4R-モナティン生成酵素を溶出させた。得られた各溶出画分について2S,4R-モナティン生成活性を測定したところ、約1.0M硫酸アンモニウム相当の画分に2S,4R-モナティン生成活性が認められた。
2S,4R-モナティン生成活性が検出された画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。得られた濃縮液を、Tris-HCl(pH7.6) 20mM、NaCl 150mMで希釈した。Tris-HCl(pH7.6) 20mM、NaCl 150mMで平衡化されたゲルろ過カラムHiLoad 16/60 Superdex 200 pg(GEヘルスケアバイオサイエンス製、CV=120ml)に供し、1ml/minの流速で溶出した。この操作により分子量約120kDaと見積もられる位置で2S,4R-モナティン生成活性が確認された。
2S,4R-モナティン生成活性が検出された画分を集めて、Tris-HCl(pH7.6) 20mM、NaCl 100mMで平衡化した陰イオン交換クロマトグラフィーカラムMono Q 5/5(ファルマシア(GEヘルスケアバイオサイエンス)製、CV=1ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 100mMで用いて洗い流した後、NaCl濃度を100mMから500mMまで直線的に変化させて、0.5ml/minの流速で吸着したタンパク質の溶出を行った。得られた各溶出画分について2S,4R-モナティン生成活性を検出したところ、約200mM NaCl相当の画分に2S,4R-モナティン生成活性が認められた。
得られた画分をSDS-PAGEに供したところ、活性画分には45kDa付近にバンドが1本認められた。このバンドを、2S,4R-モナティンを生成するアミノトランスフェラーゼの候補としてN末端アミノ酸配列解析に供した。また、内部アミノ酸配列解析に供した。
実施例2で得られた精製酵素溶液をN末端アミノ酸配列解析に供したところ、SGFTALSEAELNDLY(配列番号4)のN末端アミノ酸配列が得られた。また、SDS-PAGEゲル中の試料をトリプシン処理し(pH8.0、35℃、20時間)、逆相HPLCに供して断片ペプチドを分離した。分取した画分についてアミノ酸配列解析を行ったところ、QLDLSMGMLDVV(配列番号5)の内部アミノ酸配列が得られた。N末端アミノ酸配列、内部アミノ酸配列ともに、Bacillus pumilus SAFR-032由来アミノトランスフェラーゼ(YP_001487343)と高い相同性を示した。
実施例1と同様の方法でBacillus altitudinis AJ1616を培養した。得られた培養液から遠心分離により集菌し、ゲノムDNAを抽出した。
得られたゲノムDNAを鋳型にして、アミノトランスフェラーゼ遺伝子を含むDNA断片をPCR増幅した。プライマーは、Bacillus pumilus SAFR-032のゲノムDNA配列(CP000813)を参考にして、アミノトランスフェラーゼ遺伝子の上流300bpと、下流200bpのDNA配列から設計した、プライマーBp-u300-f(5’-ctcaggaagcaggcgcaaaaagattaattt-3’:配列番号6)およびプライマーBp-d200-r(5’-ggatgctgtctttgtcatcccaaagtggat-3’:配列番号7)を用いた。PCRは、KOD-plus-ver.2(東洋紡)を用いて以下の条件で行った。
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
実施例3で得られたN末端アミノ酸配列、内部アミノ酸配列とも一致したため、2S,4R-モナティン生成活性を有するアミノトランスフェラーゼの遺伝子を取得できたと考えられた。
(1)Bacillus altitudinis AJ1616由来アミノトランスフェラーゼ発現プラスミドの構築
Bacillus altitudinis AJ1616のゲノムDNAを鋳型にして、Bacillus altitudinis AJ1616由来アミノトランスフェラーゼ遺伝子を含むDNA断片をPCR増幅した。プライマーは、プライマー1616AT-Nde-f(5’-ggaattccatATGAGCGGTTTTACAGCGTT-3’:配列番号8)およびプライマー1616-xho-r(5’-gtcaaggagtttttctcgagTACCGTTGGTGCTGATTGAC-3’:配列番号9)を用いた。アミノトランスフェラーゼ遺伝子内のNdeI配列は、プライマー1616-delNde-f(5’-GGATTGAAGGAACAcATGAAAAAGCATGC-3’:配列番号10)およびプライマー1616-delNde-r(5’-GCATGCTTTTTCATgTGTTCCTTCAATCC-3’:配列番号11)を用いて変換した。PCRは、KOD-plus-ver.2(東洋紡)を用いて以下の条件で行った。
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
構築した発現プラスミドpET-22-1616AT-HisをE.coli BL21(DE3)に導入し、形質転換体をアンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen) 160mlに1白金耳接種し、500ml容坂口フラスコを用いて37℃で16時間振盪させた。培養終了後、得られた培養液約1000mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMにて洗浄、懸濁し、4℃で30分間超音波破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
得られた可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMで平衡化したHis-tagタンパク質精製カラムHisPrep FF 16/10(ファルマシア(GEヘルスケアバイオサイエンス)製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMで用いて洗い流した後、Imidazole濃度を20mMから250mMまで直線的に変化させて、3ml/minの流速で吸着したタンパク質の溶出を行った。
得られた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液を、Tris-HCl(pH7.6) 20mM、NaCl 100mMで希釈し、Tris-HCl(pH7.6) 20mM、NaCl 100mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 16/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 100mMで用いて洗い流した後、NaCl濃度を100mMから500mMまで直線的に変化させて、3ml/minの流速で吸着したタンパク質の溶出を行った。
各溶出画分について2S,4R-モナティン生成活性を確認し、2S,4R-モナティン生成活性が認められた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液をTris-HCl(pH7.6) 20mMで希釈し、1616AT-His溶液とした。
2S,4R-モナティンの定量は、HPLC分析により行った。分析条件は以下に示す通りである。
移動相:20mM KH2PO4/アセトニトリル=100/5
流速:1.0ml/min
カラム温度:40℃
検出:UV 280nm
カラム:CAPCELL PAK MGII、4.6×150mm、3μm(資生堂)
反応液(4R-IHOG 9.5mM、4S-IHOG 0.5mM、L-Asp 80mM、PLP 50μM、Tris-HCl 100mM、pH8.0) 0.1mlに、1616AT-Hisが0.5mg含まれるよう調製した1616AT-His溶液(実施例5)を加え、25℃で12時間反応させた。反応終了後、生成した2S,4R-モナティンを定量したところ、8.6mMであった。
反応液(IPA 50mM、PA 100mM、L-Asp 100mM、MgCl2 1mM、PLP 50μM、Tris-HCl 100mM、リン酸カリウム緩衝液 100mM、pH8.0) 0.1mlに、1616AT-Hisが0.5mg(実施例5の1616AT-His溶液を使用)、SpAld(アルドラーゼ活性を有する溶液。調製法は、下記にて詳述する。特開2006-204285号公報もまた参照)が0.01mg、Oxaloacetate Decarboxylase(シグマ、O4878)が1U含まれるよう調製し、25℃で2時間反応させた。反応終了後、生成した2S,4R-モナティンを定量したところ、5.0mMであった。
反応液(L-Trp 50mM、PA 100mM、L-Asp 400mM、MgCl2 1mM、PLP 50μM、Tris-HCl 100mM、リン酸カリウム緩衝液 100mM、pH6.5) 1.0mlに、1616AT-Hisが5mg(実施例5の1616AT-His溶液を使用)、SpAldが0.2mg、pTB2株(脱アミノ化酵素を発現し得る菌株。調製法は、下記にて詳述する。WO2009/028338もまた参照)の坂口フラスコ培養液(TB培地)が0.4ml、Superoxide Dismutase(シグマ、S8160)が200U、Oxaloacetate Decarboxylase(シグマ、O4878)が10U含まれるよう調製し、25℃で12時間反応させた。反応は試験管を用いて、140rpmで振とうさせ行った。反応終了後、生成した2S,4R-モナティンを定量したところ、22mMであった(44%の収率)。
特開2006-204285号公報、実施例5に記載のプラスミドDNA、ptrpSpALDを鋳型にして、SpAld遺伝子を含むDNA断片をPCR増幅した。プライマーは、プライマーSpAld-f-NdeI(5’-GGAATTCCATATGACCCAGACGCGCCTCAA-3’:配列番号12)およびプライマーSpAld-r-HindIII(5’-GCCCAAGCTTTCAGTACCCCGCCAGTTCGC-3’:配列番号13)を用いた。アルドラーゼ遺伝子内のE.coliレアコドン(6L-ctc、13L-ctc、18P-ccc、38P-ccc、50P-ccc、77P-ccc、81P-ccc、84R-cga)は、それぞれ6L-ctg、13L-ctg、18P-ccg、38P-ccg、50P-ccg、77P-ccg、81P-ccg、84R-cgcに変換した。6Lを変換する際には、プライマー6L-f(5’-ACCCAGACGCGCCTGAACGGCATCATCCG-3’:配列番号14)およびプライマー6L-r(5’-CGGATGATGCCGTTCAGGCGCGTCTGGGT-3’:配列番号15)を用いた。13Lを変換する際には、プライマー13L-f(5’-ATCATCCGCGCTCTGGAAGCCGGCAAGCC-3’:配列番号16)およびプライマー13L-r(5’-GGCTTGCCGGCTTCCAGAGCGCGGATGAT-3’:配列番号17)を用いた。18Pを変換する際には、プライマー18P-f(5’-GAAGCCGGCAAGCCGGCTTTCACCTGCTT-3’:配列番号18)およびプライマー18P-r(5’-AAGCAGGTGAAAGCCGGCTTGCCGGCTTC-3’:配列番号19)を用いた。38Pを変換する際には、プライマー38P-f(5’-CTGACCGATGCCCCGTATGACGGCGTGGT-3’:配列番号20)およびプライマー38P-r(5’-ACCACGCCGTCATACGGGGCATCGGTCAG-3’:配列番号21)を用いた。50Pを変換する際には、プライマー50P-f(5’-ATGGAGCACAACCCGTACGATGTCGCGGC-3’:配列番号22)およびプライマー50P-r(5’-GCCGCGACATCGTACGGGTTGTGCTCCAT-3’:配列番号23)を用いた。77P、81P、84Rを変換する際には、プライマー77P-81P-84R-f(5’-CGGTCGCGCCGTCGGTCACCCCGATCGCGCGCATCCCGGCCA-3’:配列番号24)およびプライマー77P-81P-84R-r(5’-TGGCCGGGATGCGCGCGATCGGGGTGACCGACGGCGCGACCG-3’:配列番号25)を用いた。PCRは、KOD-plus(東洋紡)を用いて以下の条件で行った。
1 cycle 94℃、2min
25 cycles 94℃、15sec
55℃、15sec
68℃、60sec
1 cycle 68℃、60sec
4℃
得られた培養液100mlから、菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mMにて洗浄、懸濁し、4℃で30分間超音波破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
上記の可溶性画分を、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 26/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=53ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mMで用いて洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、8ml/minの流速で吸着したタンパク質の溶出を行った。アルドラーゼ活性を有する画分を集めて、硫酸アンモニウム 1M、Tris-HCl(pH7.6) 20mMとなるよう、硫酸アンモニウム、Tris-HCl(pH7.6)を加えた。
反応条件:りん酸緩衝液(pH7.0)50mM、PHOG 2mM、NADH 0.25mM、MgCl2 1mM、Lactate dehydrogenase 16U/ml、25℃、340nmの吸光度を測定。
国際公開第2009/028338号、実施例2に記載のpTB2株をアンピシリン100mg/lを含むTB液体培地50mlに1白金耳接種し、500ml容坂口フラスコを用いて37℃で16時間振盪させた。得られた培養液を、pTB2株の坂口フラスコ培養液(TB培地)とした。
(1)細菌による2S,4R-モナティン合成
ニュートリエントブロス(NB)寒天培地、あるいはCM2G寒天培地(酵母エキス 10g/l、ポリペプトン 10g/l、グルコース 5g/l、NaCl 5g/l、寒天 15g/l、pH7.0)に、Rhizobium radiobacter LAT1、Rhizobium radiobacter AJ11568、Dietzia maris AJ2788、Stenotrophomonas sp. AJ3447、Stenotrophomonas sp. AJ13127、Pseudomonas chlororaphis subsp. chlororaphis NBRC3904、Micrococcus luteus NBRC3067、Stenotrophomonas sp. AJ11634、Pseudomonas putida NBRC12668、Ochrobactrum pseudogrignonense AJ3735、Stenotrophomonas sp. AJ1591、Stenotrophomonas sp. AJ3839、Brevundimonas diminuta AJ3958、Pseudomonas citronocllolis ATCC13674、Arthrobacter sp.IAM1390、Rhizobium sp. AJ12469、Rhizobium radiobacter AJ2777、Burkholderia sp. AJ3084、Microbacterium sp. AJ2787、Pseudomonas taetrolens ATCC4683、Rhizobium radiobacter ATCC4452、Rhizobium radiobacter AJ2557、Carnimonas sp. AJ3230、Rhizobium radiobacter NBRC12667、Pseudomonas fragi NBRC3458、Rhizobium radiobacter NBRC12664、Corynebacterium ammoniagenes NBRC12072、Pseudomonas sp. AJ1594、Rhizobium radiobacter ATCC6466、Pseudomonas synxantha NBRC3912、Rhizobium radiobacter ATCC4720、またはPseudomonas sp.LMG2833を塗布し、30℃で2日間培養した。
この菌体懸濁液1mlにガラスビーズ(0.1mm)を1g添加し、マルチビーズショッカー(安井器械)を用いて菌体を破砕した。得られた破砕液を遠心分離し、上清を菌体抽出液とした。
実施例1と同様にして、2S,4R-モナティン合成反応および2S,4R-モナティンの定量を行ったところ、2S,4R-モナティンの生成量は以下の通りであった(表2)。
YMPG寒天培地(酵母エキス 3g/l、麦芽エキス 3g/l、ポリペプトン 5g/l、グルコース 10g/l、寒天 15g/l、pH7.0)にNocardia globerula ATCC21022を塗布し、30℃で2日間培養した。
得られた菌体をYMPG培地(酵母エキス 3g/l、麦芽エキス 3g/l、ポリペプトン 5g/l、グルコース 10g/l、pH7.0) 3mlに一白金耳接種し、試験管で30℃にて16時間振とう培養した。培養液2mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mMにて洗浄、懸濁し、菌体懸濁液を1ml調製した。
この菌体懸濁液1mlにガラスビーズ(0.1mm)を1g添加し、マルチビーズショッカー(安井器械)を用いて菌体を破砕した。得られた破砕液を遠心分離し、上清を菌体抽出液とした。
実施例1と同様にして、2S,4R-モナティン合成反応および2S,4R-モナティンの定量を行ったところ、2S,4R-モナティンの生成量は以下の通りであった(表3)。
YPD寒天培地(酵母エキス 10g/l、ポリペプトン 20g/l、グルコース 20g/l、寒天 15g/l)にLodderomyces elongisporus CBS2605、Candida norvegensis NBRC0970、Candida inconspicua NBRC0621、Yarrowia lypolytica NBRC0746を塗布し、30℃で2日間培養した。
得られた菌体をYPD培地(酵母エキス 10g/l、ポリペプトン 20g/l、グルコース 20g/l) 3mlに一白金耳接種し、試験管で30℃にて16時間振とう培養した。培養液2mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mMにて洗浄、懸濁し、菌体懸濁液を1ml調製した。
この菌体懸濁液1mlにガラスビーズ(0.5mm)を1g添加し、マルチビーズショッカー(安井器械)を用いて菌体を破砕した。得られた破砕液を遠心分離し、上清を菌体抽出液とした。
実施例1と同様にして、2S,4R-モナティン合成反応および2S,4R-モナティンの定量を行ったところ、2S,4R-モナティンの生成量は以下の通りであった(表4)。
還元反応濃縮液101.40g(モナティン36.62g、125.28ミリモル含有、(2S,4R):(2R,4R)=32:68)にエタノール149.00gを加えた後、種晶として2R,4R-モナティンカリウム塩一水和物0.25gを添加し、56℃、4時間攪拌し、2R,4R-モナティンカリウム塩一水和物の優先晶析を行った。析出した結晶をろ過により分離し(湿結晶31.27g)、母液225.80gを得た(モナティン22.41g、76.68ミリモル含有、(2S,4R):(2R,4R)=53:47)。この母液を10℃に冷却した後、5時間攪拌し、2S,4R-モナティンカリウム塩二水和物の晶析を行った。析出した結晶をろ過により分離し(湿結晶32.74g)、減圧乾燥後、目的とする2S,4R-モナティンカリウム塩二水和物9.88g(15.68ミリモル)を得た(HPLC純度:55.5%)。この粗結晶9.35gを水25.37gに溶解し、その溶解液にエタノール58.99gを加え25℃にて5時間攪拌し、2S,4R-モナティンカリウム塩二水和物の精晶析を行った。析出した結晶をろ過により分離し(湿結晶4.49g)、減圧乾燥後、目的とする2S,4R-モナティンカリウム塩二水和物3.75g(9.62ミリモル)を得た(HPLC純度:96.0%)。
測定装置:平沼水分自動測定装置 AQV-2000 (平沼産業(株)製)
測定条件:滴定液=Hydranal Composite 5K(Riedel de Haen社製)
装置:東ソーIC2001
カラム:TSKgel SuperIC-Cation(4.6×150mm)
ガードカラム:TSKgel SuperIC-Cation(1cm)
サプレスゲル:TSKgel TSKsuppressIC-C
カラム温度:40℃
溶離液流量:0.7ml/min.
サンプル注入量:30μl
検出:電気伝導度
溶離液組成:2.2mMメタンスルホン酸+1.0mM18-クラウン-6ーエーテル+0.5mMヒスチジン混合水溶液
ESI-MS計算値 C14H16N2O5=292.11
ESI-MS分析値 C14H16N2O5=290.9[M-H]-
2S,4R-モナティンカリウム塩二水和物0.15g(0.38ミリモル)を70%エタノール水溶液10.0gに添加し、60℃にて完全に溶解させた。その溶解液に5‐ニトロサリチルアルデヒド7.6mg(0.045ミリモル)、酢酸7.5μL(0.13ミリモル)を添加し60℃にて48時間攪拌した。反応液をHPLCにて分析し、定量を行ったところ、反応液中の2S,4R-モナティンと2R,4R-モナティンのモル比は、1:2.1であった。
2S,4R-モナティンカリウム塩二水和物0.15g(0.38ミリモル)を70%エタノール水溶液10.0gに添加し、60℃にて完全に溶解させた。その溶解液にピリドキサール塩酸塩9.1mg(0.045ミリモル)、酢酸7.5μL(0.13ミリモル)を添加し60℃にて48時間攪拌した。反応液をHPLCにて分析し、定量を行ったところ、反応液中の2S,4R-モナティンと2R,4R-モナティンのモル比は、1:1.3であった。
2S,4R-モナティンカリウム塩二水和物0.15g(0.38ミリモル)を70%エタノール水溶液10.0gに添加し、60℃にて完全に溶解させた。その溶解液に5‐リン酸ピリドキサール一水和物12.8mg(0.048ミリモル)、酢酸7.5μL(0.13ミリモル)を添加し60℃にて48時間攪拌した。反応液をHPLCにて分析し、定量を行ったところ、応液中の2S,4R-モナティンと2R,4R-モナティンのモル比は、1:1.1であった。
2S,4R-モナティンカリウム塩二水和物0.15g(0.38ミリモル)を70%エタノール水溶液10.0gに添加し、60℃にて完全に溶解させた。その溶解液にサリチルアルデヒド5.3mg(4.6μL,0.043ミリモル)、酢酸7.5μL(0.13ミリモル)を添加し60℃にて48時間攪拌した。反応液をHPLCにて分析し、定量を行ったところ、反応液中の2S,4R-モナティンと2R,4R-モナティンのモル比は、1:0.6であった。
2S,4R-モナティンカリウム塩二水和物0.15g(0.38ミリモル)を70%エタノール水溶液10.0gに添加し、60℃にて完全に溶解させた。その溶解液に3,5-ジクロロサリチルアルデヒド8.1mg(0.042ミリモル)、酢酸7.5μL(0.13ミリモル)を添加し60℃にて48時間攪拌した。反応液をHPLCにて分析し、定量を行ったところ、反応液中の2S,4R-モナティンと2R,4R-モナティンのモル比は、1:1.5であった。
2S,4R-モナティンカリウム塩二水和物を20%エタノール水溶液に添加し、60℃にて完全に溶解させる。この溶解液に5-ニトロサリチルアルデヒドを2S,4R-モナティンに対して5モル%、酢酸を2S,4R-モナティンに対して30モル%添加し、48時間攪拌する。この反応液(2S,4R-モナティン:2R,4R-モナティン=1:2.1)にエタノール濃度が70%となるようエタノールを添加した後、種晶として2R,4R-モナティンカリウム塩一水和物を反応液中の2R,4R-モナティンに対して1%添加し、60℃、48時間攪拌し、異性化晶析を行う。析出した結晶をろ過により分離し、減圧乾燥後、目的とする2R,4R-モナティンカリウム塩一水和物を得る。
2S,4R-モナティンカリウム塩二水和物0.15g(0.38ミリモル)を70%エタノール水溶液10.0gに添加し、60℃にて完全に溶解させた。その溶解液にグリオキシル酸5.1mg(0.069ミリモル)、酢酸7.5μL(0.13ミリモル)を添加し60℃にて48時間攪拌した。反応液をHPLCにて分析し、定量を行ったところ、反応液中の2S,4R-モナティンと2R,4R-モナティンのモル比は、1:0.07であった。
(1)部位特異的変異による変異型LAT発現プラスミドの作製
部位特異的変異による変異型AJ1616株由来LAT発現プラスミドの作製は、Stratagene社製QuikChange Site-Directed Mutagenesis Kitのプロトコルに準拠して行った。目的とするヌクレオチド残基の変異(置換)を導入し、かつ2本鎖DNAの各々の鎖に相補的になるように設計した1セットのDNAプライマーを合成した。作製した変異体と作製に用いたプライマーのヌクレオチド配列をそれぞれ表5、6に示す。pET22-AJ1616LAT-His(C)を鋳型とし、以下のPCR条件で変異型プラスミドを作製した。
1 cycle 95℃ 1 min
18 cycles 95℃ 30 sec
55℃ 1 min
68℃ 8 min
cycles終了後 4℃
得られた変異型AJ1616LAT発現プラスミドでE.coli JM109(DE3)を形質転換し、変異型AJ1616LAT発現株を作製した。LB-amp(100mg/l)プレート上で生育させた変異型AJ1616LAT発現株、pET22-AJ1616LATmut-His(C)/E.coli JM109(DE3)の菌体を、アンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen)100mlに植菌し、37℃で16時間、坂口フラスコを用いて振とう培養を行った。培養終了後、得られた培養液より菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMにて洗浄、懸濁し、超音波破砕を行った。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。得られた可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMで平衡化したHis-tagタンパク質精製カラムHis TALON superflow 5ml Cartridge(Clontech)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMを用いて洗い流した後、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 150mMを用いて、5ml/minの流速で吸着したタンパク質の溶出を行った。得られた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液をTris-HCl(pH7.6) 20mMで希釈し、変異型AJ1616LAT溶液とした。必要に応じて、培養液量、His TALONカラムの連結数を増やして精製を行った。
タンパク質の濃度の測定は、ナカライテスク社のプロテインアッセイCBB溶液(5倍希釈して使用)を用いて行った。タンパク質の濃度は、0.05、0.1、0.25、0.5mg/mlのBSA溶液をスタンダートとして検量線を作成することにより、算出した。
AJ1616LATの各種基質に対する活性測定を実施した。アミノ基転移反応のアミノドナー基質としては100mM L-Aspを用い、10mMの各種ケト酸に対する比活性を比色法で測定した。
L-Asp/α-KG(α-ケトグルタル酸)活性:L-Asp-Na 100mM、α-KG-2Na 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM、NADH 0.25mM、MDH 2U/ml、25℃。340nmの減少から活性を算出。MDHはMalic Dehydrogenase from porcine heart(Sigma)を用いた。なお、L-Asp/α-KG活性は、表9において、アミノトランスフェラーゼ活性の項目「α-KG」に示す。
L-Asp/PA活性:L-Asp-Na 100mM、PA-Na 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM、NADH 0.25mM、MDH(上記と同様) 2U/ml、25℃。340nmの減少から活性を算出。なお、L-Asp/PA活性は、表9において、アミノトランスフェラーゼ活性の項目「PA」に示す。
L-Asp/(±)-MHOG(4-hydroxy-4-methyl-2-ketoglutarate)活性:L-Asp-Na 100mM、(±)-MHOG 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM、NADH 0.25mM、MDH 2U/ml、LDH 10U/ml、25℃。340nmの減少から活性を算出。LDHはD-Lactate dehydrogenase from Leuconostoc mesenteroides(オリエンタル酵母)を用いた。(±)-MHOGに微量に混在しているPAを除くため、LDHを添加した。なお、L-Asp/(±)-MHOG活性は、表9において、アミノトランスフェラーゼ活性の項目「±MHOG」に示す。
目的活性である4R-IHOGからの2S,4R-モナティン生成活性及びIPAからのL-Trp副生活性をそれぞれ測定した。アミノ基転移反応のアミノドナー基質としては100mM L-Aspを用い、10mMの各種ケト酸に対するアミノ基転移反応を行い、生成したアミノ酸量をUPLCないしHPLCで定量し、比活性を算出した。
L-Asp/4R-IHOG活性(10mM):L-Asp-Na 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃。UPLC分析を行い、生成した2S,4R-モナティン、2S,4S-モナティンを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/4R-IHOG活性は、表9において、アミノトランスフェラーゼ活性の項目「4R-IHOG」に示す。
L-Asp/IPA活性:L-Asp-Na 100mM、IPA 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM(pHは反応液作成後に1N NaOHで8.0に調整)、25℃。UPLC分析を行い、生成したTrpを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/IPA活性は、表9において、アミノトランスフェラーゼ活性の項目「IPA」に示す。
生成したモナティン及びTrpの定量はWaters社製 ACQUITY UPLCシステムを用いて定量した。測定条件を以下に示す。0.2mlで15分間反応を行ってから反応停止し、反応停止後の反応液を遠心し上清0.2ml程度をUPLC分析に供した。Sample-Blankが0.01-0.05mMの範囲に入る希釈段階で測定した結果を活性値として採用した。
HPLC条件(モナティン、Trp、IPA、IAA(インドール酢酸)、IAD(インドールアルデヒド)定量用条件)
カラム:CAPCELL PAK C18 TYPE MGII 3μm 4.6mm x 150mm(資生堂)
カラム温度:40℃
検出波長:280nm
流速:1.0ml/min
移動層:A:20mM KH2PO4/CH3CN=100/5、B:CH3CN
作製した変異体を用いて100mM L-Aspをアミノドナーとした際の10mM ケト酸に対する比活性測定結果を表9に示す。作製した変異体のいずれにおいても、目的とする4R-IHOGを基質とした2S,4R-モナティン生成活性の向上が認められた。また、目的活性に対する副反応の相対値に関しても、いずれの変異体においても目的活性(2S,4R-モナティン生成活性)に対するL-Trp副生活性、MHG(4-hydroxy-4-methyl glutamate)副生活性、L-Ala副生活性の低下が認められた。
E.coli JM109ΔaspCは、以下の方法で構築した。E.coli JM109/pKD46をLB-amp(100mg/l)プレート上で30℃、一晩培養した。得られた菌体をLB(Amp 100mg/l、L-Arabinose 10mMを含む)50mlに接種した。これを30℃で坂口フラスコを用いて振とう培養し、O.D.610が0.6程度になったところで培養温度を37℃に変更し、さらに1時間振とう培養を行った。得られた培養液から遠心分離で集菌、10% Glycerolで洗浄し、再度遠心分離、集菌した。これを10% Glycerolで懸濁しコンピテントセルとした。
pMW118-attL-cat-attRを鋳型にして、プライマーaspC-L1(5’-TTTGAGAACATTACCGCCGCTCCTGCCGACCCGATTCTGGGCtgaagcctgcttttttat-3’:配列番号36)とプライマーaspC-R1(5’-CAGCACTGCCACAATCGCTTCGCACAGCGGAGCCATGTTATCcgctcaagttagtataaa-3:配列番号37)を用いてPCR増幅を行った。得られたPCR産物をアガロースから抽出し、aspC遺伝子破壊用DNA断片とした。PCRは、KOD-plus-ver.2(東洋紡)を用いて行った。
精製したDNA断片でコンピテントセルを形質転換し、目的の形質転換体を37℃、LB-Cm(20mg/l)プレート上で選択した。形質転換体のaspC遺伝子領域にattL-cat-attRが挿入されていることをコロニーPCRで確認した。用いたプライマーは、プライマーaspC-up(5’-AACCTCTTGGCAACGGTAAAAAAGCTGAAC-3’:配列番号38)とプライマーattL-1(5’-TAGTGACCTGTTCGTTGC-3’:配列番号39)、プライマーaspC-down(5’-GCCTGCGCAAAGTCGTATGTTTGGTCTGGA-5’:配列番号40)とプライマーattR-1(5’-TTACGTTTCTCGTTCAGC-3’:配列番号41)である。PCRは、Z-taq(TAKARA)を用いた。
得られた形質転換体をLB(Cm 20mg/l)3mlに接種し、37℃で6時間振とう培養した。得られた培養液から遠心分離で集菌、10% Glycerolで洗浄し、再度遠心分離、集菌した。これを10% Glycerolで懸濁しコンピテントセルとした。
ゲノムDNAに挿入されたCm耐性遺伝子配列を除去するため、pMW-intxis-tsで上記コンピテントセルを形質転換した。目的の形質転換体を30℃、LB-amp(100mg/l)プレート上で選択した。得られた形質転換体を42℃、LBプレート上で一晩培養し、次に菌体をLB-amp(100mg/l)プレート、LB-Cm(20mg/l)プレートにそれぞれストリークし37℃で培養した。Amp、Cmどちらのプレートでも生育しないことを確認し、さらにプライマーaspC-up(5’-AACCTCTTGGCAACGGTAAAAAAGCTGAAC-3’:配列番号38)とプライマーaspC-down(5’-GCCTGCGCAAAGTCGTATGTTTGGTCTGGA-5’:配列番号40)を用いたコロニーPCRによりCm耐性遺伝子の除去を確認した。PCRは、Z-taq(TAKARA)を用いた。
得られた株をaspC欠損株、E.coli JM109ΔaspCとした。また、デアミナーゼ発現株pTB2/E.coli JM109ΔaspCは、デアミナーゼ発現プラスミドpTB2でE.coli JM109ΔaspCを形質転換し、構築した。本菌株をLB-amp(100mg/l)プレート上で37℃、一晩培養した。得られた菌体をTB-amp(100mg/l)100mlに植菌、坂口フラスコを用いて37℃で16時間振とう培養を行った。得られた培養液をPs_aad brothとして用いた。
Pseudomonas putida KT2440株由来OAA Decarboxylaseの遺伝子合成をGenScript社に依頼し、OAA Decarboxylase遺伝子を含むDNA断片がpUC57に挿入されたプラスミドDNAを得た。コドン使用頻度はE.coliでの発現用に至適化された(配列番号42、43を参照)。このプラスミドをNdeI、XhoIで切断し、NdeI、XhoIで切断したpET22bに挿入、得られたプラスミドをpET22-PpODC-His(C)と命名した。得られたプラスミドでE.coli BL21(DE3)を形質転換し、PpODC-His(C)の発現株pET22-PpODC-His(C)/E.coli BL21(DE3)を得た。LB-amp(100mg/l)プレート上で生育させたPpODC-His(C)発現株、pET22-PpODC-His(C)/E.coli BL21(DE3)の菌体を、アンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen)100mlに植菌し、30℃で16時間、坂口フラスコを用いて振とう培養を行った。培養終了後、得られた培養液より菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMにて洗浄、懸濁し、超音波破砕を行った。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。得られた可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMで平衡化したHis-tagタンパク質精製カラムHis TALON superflow 5ml Cartridge(Clontech)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMを用いて洗い流した後、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 150mMを用いて、5ml/minの流速で吸着したタンパク質の溶出を行った。得られた画分を集めて、アミコン ウルトラ-15 10k(ミリポア)を用いて濃縮した。濃縮液をTris-HCl (pH7.6) 20mMで希釈し、PpODC溶液とした。
ODC活性の測定条件は以下に示す条件で行った。
OAA 10 mM、Tris-HCl(pH8.0) 100mM、NADH 0.25mM、LDH 10U/ml、25℃。340nmの減少から活性を算出した。LDHはD-Lactate dehydrogenase from Leuconostoc mesenteroides(オリエンタル酵母)を用いた。1 mlスケールで反応、分析を行い測定結果(SampleΔ340 nm/min)-(BlankΔ340nm/min)の値が0.05-0.15の範囲に入る希釈段階での活性値を採用した。酵素の希釈にはTris-HCl(pH7.6) 20mM、BSA 0.01%を用いた。
精製した変異型AJ1616LATを用いて、以下の条件で22時間反応を行った。反応は試験管を用いて1mlで行った。サンプリングは14、18、22時間後に行った。サンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL 遠心式フィルター10kDaを用いて限外ろ過、ろ液を分析した。分析にはHPLCを用いた。
Ps_aadブロス:実施例17に記載の方法に従って調製した。
SpAld精製酵素:実施例6に記載の方法に従ってSpALD発現株のジャー培養を行い、さらに60℃で1時間の熱処理を行った。得られた熱処理後の培養液100mlから、菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mMにて洗浄、懸濁し、超音波破砕を行った。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。この可溶性画分が硫酸アンモニウム 1M、Tris-HCl(pH7.6) 20mMを含むよう、硫酸アンモニウム、Tris-HCl(pH7.6)を添加した。この溶液を硫酸アンモニウム 1M、Tris-HCl(pH7.6) 20mMで平衡化した疎水性クロマトグラフィーカラムHiLoad 26/10 Phenyl Sepharose HP(GEヘルスケアバイオサイエンス社製、CV=53ml)カラムに供して担体に吸着させた。担体に吸着しなかった非吸着タンパク質を硫酸アンモニウム 1M、Tris-HCl(pH7.6) 20mMを用いて洗い流した後、硫酸アンモニウム濃度を1Mから0Mまで直線的に変化させて、8ml/minの流速で吸着したタンパク質の溶出を行った。活性が検出された画分を集めて、アミコン ウルトラ-15 10k(ミリポア)を用いて濃縮した。得られた濃縮液を、Tris-HCl(pH7.6) 20mMで希釈し、SpAld溶液とした。アルドラーゼ活性の測定には、PHOG分解活性測定法を用いた(PHOG 2mM、KPB 50mM、MgCl2 1mM、NADH 0.25mM、LDH 16U/ml、25℃、pH7.0、340nmの減少から活性を算出)。LDHはD-Lactate dehydrogenase from Leuconostoc mesenteroides(オリエンタル酵母)を用いた。
変異型AJ1616LAT:LB-amp(100mg/l)プレート上で生育させた変異型AJ1616LAT発現株、pET22-AJ1616LATmut-His(C)/E.coli JM109(DE3)の菌体を、アンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen)100mlに植菌し、37℃で16時間、坂口フラスコを用いて振とう培養を行った。培養終了後、得られた培養液より菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMにて洗浄、懸濁し、超音波破砕を行った。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。得られた可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMで平衡化したHis-tagタンパク質精製カラムHis TALON superflow 5ml Cartridge(Clontech)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl (pH7.6) 20mM、NaCl 300mM、Imidazole 10mMを用いて洗い流した後、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 150mMを用いて、5ml/minの流速で吸着したタンパク質の溶出を行った。得られた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液をTris-HCl(pH7.6) 20mMで希釈し、変異型AJ1616LAT溶液とした。必要に応じて、培養液量、His TALONカラムの連結数を増やして精製を行った。
OAA DCase:Oxaloacetate Decarboxylase from Pseudomonas sp.(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
SOD:Superoxide Dismutase from bovine liver(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
精製したAJ1616LAT-ID166を用いて、以下の条件で6時間反応を行った。反応は1 L容S-ジャーを用いて400mlで行った。適宜サンプリングを行い、サンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL 遠心式フィルター 10kDaを用いて限外ろ過、ろ液を分析した。分析にはHPLCおよびキャピラリー電気泳動を用いた。
精製したAJ1616LAT-ID189を用いて、以下の条件で27時間反応を行った。反応は250mL容S-ジャーを用いて80mlで行った。適宜サンプリングを行い、サンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL 遠心式フィルター 10kDaを用いて限外ろ過、ろ液を分析した。分析にはHPLCおよびキャピラリー電気泳動を用いた。
Ps_aadブロスはpTB2/E.coli JM109ΔaspCブロスを用いた。SpAldブロスは実施例19に記載の熱処理ブロスを用いた。PpODCは実施例18に記載の精製酵素を用いた。SODはSuperoxide Dismutase from bovine liver(Sigma)を用いた。
実施例20の酵素反応液435.45g(ロット101213 J4)をUF(MWCO:3000)処理して得られた透過液435.66gに、ZN炭2.59gを添加し、室温(約26℃)で1時間撹拌した。活性炭を桐山濾紙(5C)で濾過し、得られた濾過液を1L四つ口フラスコに移液した。そのフラスコを5℃の恒温槽に浸漬し、35%塩酸でpHを3.5になるように中和し、メカニカルスタラーで撹拌した(120rpm)。種晶を48mg添加し、結晶が析出しはじめると溶液pHが上昇するので、目標pHを維持するようにpHコントロラーとペリスターポンプを用いて1N塩酸を逐次添加した。24時間撹拌して得られたスラリー溶液を濾過し、水10mLで結晶洗浄を行った後、湿結晶を40℃で減圧乾燥して2S,4R-モナティン 6.81gを取得した。得られた結晶をHPLC及び1H-NMR分析を行いその品質を確認した。
1H-NMR(in D2O+K2CO3)
2.08-2.14(1H,dd),2.35-2.39(1H,dd),3.09-3.17(2H,dd),3.85-3.88(1H,dd),7.04-7.15(3H,m),7.39-7.41(1H,m),7.64-7.66(1H,d)
水 3.27gに実施例22で取得した2S,4R-モナティン 3.10g(10.4mmol)及び50%KOH 1.165g(10.4mmol)を溶解し、更にEtOH 1.3g、5-ニトロサリチルアルデヒド0.0869g(0.052mmol)及び酢酸0.187g(3.12mmol)を添加した。25時間後にEtOH 20.5g及び種晶(2R、4R-モナティン) 10mgを添加し、更に46.5時間撹拌した。得られたスラリー溶液を室温まで冷却してから濾過を行い、85%EtOH水 4gで結晶洗浄を行った後、湿結晶を40℃で減圧乾燥して粗2R,4R-モナティン2.3gを取得した。得られた粗2R,4R-モナティン 2.1gを水6mLに溶解しBA炭0.2gを添加して室温(25℃前後)で1時間撹拌後、0.45umのメンブレンフィルターで処理液を濾過し、その濾過液を6.38gまで減圧濃縮した。45℃で濃縮液にEtOH 12gを滴下し1時間撹拌した。更にEtOH 13.5gを1時間で定量滴下し、45℃で16時間撹拌後、25℃まで冷却した。得られたスラリー液を濾過し、85%EtOH水 3gで結晶洗浄を行った後、湿結晶を40℃で減圧乾燥して2R,4R-モナティン 1.9g(5.46mmol)を取得した。得られた結晶、母液、洗液をHPLCで分析し、収率及び品質解析を実施した。
1H-NMR(in D2O)
1.93-2.00(1H,dd),2.57-2.61(1H,dd),2.99-3.02(1H,d),3.19-3.22(1H,d)3.55-3.56(1H,dd),7.04-7.15(3H,m),7.39-7.41(1H,m),7.64-7.66(1H,d)
精製したAJ1616LAT-ID296を用いて、以下の条件で51時間反応を行った。反応は250ml容ジャーを用いて80mlで行った。適宜サンプリングを行い、サンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL遠心式フィルター 10kDaを用いて限外ろ過、ろ液を分析した。分析にはHPLCを用いた。
Ps_aad ブロスはpTB2/E.coli JM109ΔaspCブロスを用いた。SpAldブロスは実施例19に記載の熱処理ブロスを用いた。PpODCは実施例18に記載の精製酵素を用いた。SODはSuperoxide Dismutase from bovine liver(Sigma)を用いた。
Rhizobium radiobacter AJ3976の可溶性画分から、2S,4R-モナティンを生成するアミノトランスフェラーゼの精製を以下の通り行った。L-Asp-Na-1aq 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃で反応を行い、生成した2S,4R-モナティンをUPLC分析で定量した。
LB寒天培地にRhizobium radiobacter AJ3976を塗布し、30℃で2日間培養した。
得られた菌体を酵素生産培地(酵母エキス 10g/l、トリプトン 10g/l、グルコース 1g/l、リン酸水素二カリウム 3g/l、リン酸二水素カリウム 1g/l、硫酸マグネシウム七水和物 0.1g/l、硫酸アンモニウム 5g/l)160mlに一白金耳接種し、500ml容坂口フラスコで30℃にて20時間振とう培養した。得られた培養液約1920mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mMにて洗浄、懸濁し、4℃で30分間超音波破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
上記の可溶性画分を、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 26/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=53ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mMで洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、2ml/minの流速で吸着したタンパク質の溶出を行った。各溶出画分について2S,4R-モナティン生成活性を検出したところ、約250mM NaCl相当の画分に2S,4R-モナティン生成活性が認められた。
2S,4R-モナティン生成活性が検出された画分を集めて、硫酸アンモニウム 1.0M、Tris-HCl 20mM(pH7.6)となるよう、硫酸アンモニウム、Tris-HCl(pH7.6)を添加した。この溶液を硫酸アンモニウム 1.0M、Tris-HCl 20mM(pH7.6)で平衡化した疎水性クロマトグラフィーカラムHiLoad 16/10 Phenyl Sepharose HP(GEヘルスケアバイオサイエンス社製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかった非吸着タンパク質を硫酸アンモニウム1.0M、Tris-HCl(pH7.6) 20mMを用いて洗い流した後、硫酸アンモニウム濃度を1.0Mから0Mまで直線的に変化させて、3ml/minの流速で2S,4R-モナティン生成酵素を溶出させた。得られた各溶出画分について2S,4R-モナティン生成活性を測定したところ、約0.9M硫酸アンモニウム相当の画分に2S,4R-モナティン生成活性が認められた。
2S,4R-モナティン生成活性が検出された画分を集めて、アミコン ウルトラ-15 10k(ミリポア)を用いて濃縮した。得られた濃縮液を、Tris-HCl(pH7.6) 20mM、NaCl 150mMで希釈した。Tris-HCl(pH7.6) 20mM、NaCl 150mMで平衡化されたゲルろ過カラムHiLoad 16/60 Superdex 200 pg(GEヘルスケアバイオサイエンス製、CV=120ml)に供し、1ml/minの流速で溶出した。この操作により分子量約100kDaと見積もられる位置で2S,4R-モナティン生成活性が確認された。
得られた画分をSDS-PAGEに供したところ、活性画分には47kDa付近にバンドが1本認められた。このバンドを、2S,4R-モナティンを生成するアミノトランスフェラーゼの候補としてN末端アミノ酸配列解析に供した。
実施例25で得られた精製酵素溶液をN末端アミノ酸配列解析に供したところ、AFLADILSRVKPSATIAVTQ(配列番号44)のN末端アミノ酸配列が得られた。N末端アミノ酸配列は、Agrobacterium tumefaciens str.C58由来アスパラギン酸アミノトランスフェラーゼ(AAK87940)と高い相同性を示した。
実施例25と同様の方法でRhizobium radiobacter AJ3976を培養した。得られた培養液から遠心分離により集菌し、ゲノムDNAを抽出した。
得られたゲノムDNAを鋳型にして、アミノトランスフェラーゼ遺伝子を含むDNA断片をPCR増幅した。プライマーは、Agrobacterium tumefaciens str.C58のゲノムDNA配列を参考にして、アミノトランスフェラーゼ遺伝子の上流100bpと、下流100bpのDNA配列から設計した、プライマーAg-u100-f(5’-ctggtgcagataagccggcttttgacc-3’:配列番号45)およびプライマーAg-d100-r(5’-ccaccttcatcatgctgctgtttctcg-3’:配列番号46)を用いた。PCRは、KOD-plus-ver.2(東洋紡)を用いて以下の条件で行った。
1 cycle 94℃、2min
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
実施例26で得られたN末端アミノ酸配列とも一致したため、2S,4R-モナティン生成活性を有するアミノトランスフェラーゼの遺伝子を取得できたと考えられた。
(1)Rhizobium radiobacter AJ3976由来アミノトランスフェラーゼ発現プラスミドの構築
Rhizobium radiobacter AJ3976のゲノムDNAを鋳型にして、Rhizobium radiobacter AJ3976由来アミノトランスフェラーゼ遺伝子を含むDNA断片をPCR増幅した。プライマーは、プライマー3976AT-Nde-f(5’-ggaattccatATGGCCTTCCTTGCCGACATTCTCT-3’:配列番号49)およびプライマー3976-xho-r(5’-actccgctcgagACGGCAATCGGCGCAGAAACGCTGA-3’:配列番号50)を用いた。PCRは、KOD-plus-ver.2(東洋紡)を用いて以下の条件で行った。
1 cycle 94℃、2min
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
構築した発現プラスミドpET-22-3976AT-HisをE.coli BL21(DE3)に導入し、形質転換体をアンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen) 160mlに白金耳接種し、500ml容坂口フラスコを用いて37℃で16時間振盪させた。培養終了後、得られた培養液約1000mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMにて洗浄、懸濁し、4℃で30分間超音波破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
得られた可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMで平衡化したHis-tagタンパク質精製カラムHisPrep FF 16/10(ファルマシア(GEヘルスケアバイオサイエンス)製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMで用いて洗い流した後、Imidazole濃度を20mMから250mMまで直線的に変化させて、3ml/minの流速で吸着したタンパク質の溶出を行った。
得られた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液を、Tris-HCl(pH7.6) 20mMで希釈し、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 16/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mMで用いて洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、3ml/minの流速で吸着したタンパク質の溶出を行った。
各溶出画分について2S,4R-モナティン生成活性を確認し、2S,4R-モナティン生成活性が認められた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液をTris-HCl(pH7.6) 20mMで希釈し、3976AT-His溶液とした。
(1)比色法でのL-Asp/α-KG、L-Asp/PA、およびL-Asp/(±)-MHOG活性測定
AJ3976LATの各種基質に対する活性測定を実施した。アミノ基転移反応のアミノドナー基質としては100mM L-Aspを用い、10mMの各種ケト酸に対する比活性を比色法で測定した。
L-Asp/α-KG活性:L-Asp-Na-1aq 100mM、α-KG-2Na 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH 2U/ml、25℃。340nmの減少から活性を算出。MDHはMalic Dehydrogenase from porcine heart(Sigma)を用いた。なお、L-Asp/α-KG活性は、表13において、アミノトランスフェラーゼ活性の項目「α-KG」に示す。
L-Asp/PA活性:L-Asp-Na-1aq 100mM、PA-Na 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH(上記と同様)2U/ml、25℃。340nmの減少から活性を算出。なお、L-Asp/PA活性は、表13において、アミノトランスフェラーゼ活性の項目「PA」に示す。
L-Asp/(±)-MHOG活性:L-Asp-Na-1aq 100mM、(±)-MHOG 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH(上記と同様)2U/ml、LDH 10U/ml、25℃。340nmの減少から活性を算出。LDHはD-Lactate dehydrogenase from Leuconostoc mesenteroides(オリエンタル酵母)を用いた。(±)-MHOGに微量に混在しているPAを除くため、LDHを添加した。なお、L-Asp/(±)-MHOG活性は、表13において、アミノトランスフェラーゼ活性の項目「±MHOG」に示す。
目的活性である4R-IHOGからの2S,4R-モナティン生成活性、(±)-IHOGからの2S,4R-モナティン及び2S,4S-モナティン生成活性、及びIPAからのL-Trp副生活性をそれぞれ測定した。アミノ基転移反応のアミノドナー基質としては100mM L-Aspを用い、10mMの各種ケト酸に対するアミノ基転移反応を行い、生成したアミノ酸量をUPLCで定量し、比活性を算出した。
L-Asp/4R-IHOG活性:L-Asp-Na-1aq 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃。UPLC分析を行い、生成した2S,4R-モナティン、2S,4S-モナティンを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/4R-IHOG活性は、表13において、アミノトランスフェラーゼ活性の項目「4R-IHOG」に示す。
L-Asp/(±)-IHOG活性:L-Asp-Na-1aq 100mM、(±)-IHOG 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃。UPLC分析を行い、生成した2S,4R-モナティン、2S,4S-モナティンを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/4R-IHOG活性は、表13において、アミノトランスフェラーゼ活性の項目「±IHOG」に示す。
L-Asp/IPA活性:L-Asp-Na-1aq 100mM、IPA 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM(pHは反応液作成後に1N NaOHで8.0に調整)、25℃。UPLC分析を行い、生成したTrpを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/IPA活性は、表13において、アミノトランスフェラーゼ活性の項目「IPA」に示す。
生成したモナティン及びTrpの定量はWaters社製 ACQUITY UPLCシステムを用いて定量した。測定条件を以下に示す。0.2mlで15分間反応を行ってから反応停止し、反応停止後の反応液を遠心し上清0.2ml程度をUPLC分析に供した。
3976-AT-Hisを用いて100mM L-Aspをアミノドナーとした際の10mM ケト酸に対する比活性測定結果を表13に示す。
実施例28で作製したpET-22-3976AT-His/E.coli BL21(DE3)をアンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen)3mlに白金耳接種し、試験管を用いて37℃で16時間振盪させた。培養終了後、得られた培養液1mlより菌体を遠心分離により集め、1 mlのBugBuster Master Mix (Novagen)に懸濁した。得られた懸濁液を室温にて15分間静置して溶菌させ、遠心分離により菌体残渣を除き、得られた上清を可溶性画分とした。
得られた可溶性画分を用いて4R-IHOGからの2S,4R-モナティン合成反応を行った。反応液〔L-Asp-Na-1aq 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl 100mM、pH8.0〕 0.1mlに、上記の可溶性画分が0.05ml含まれるように加え、25℃で1時間反応させた。反応終了後、生成した2S,4R-モナティンを定量したところ、0.84mMであった。2S,4R-モナティンの定量は、UPLC分析により行った。分析条件は実施例29と同様である。
Rhizobium sp.AJ12469の可溶性画分から、2S,4R-モナティンを生成するアミノトランスフェラーゼの精製を以下の通り行った。実施例25と同様にして、2S,4R-モナティン合成反応および2S,4R-モナティンの定量を行った。
LB寒天培地にRhizobium sp.AJ12469を塗布し、30℃で2日間培養した。
得られた菌体を酵素生産培地(酵母エキス 10g/l、トリプトン 10g/l、グルコース 1g/l、リン酸水素二カリウム 3g/l、リン酸二水素カリウム 1g/l、硫酸マグネシウム七水和物 0.1g/l、硫酸アンモニウム 5g/l)160mlに一白金耳接種し、500ml容坂口フラスコで30℃にて16時間振とう培養した。得られた培養液約1920mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mMにて洗浄、懸濁し、4℃で30分間超音波破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
上記の可溶性画分を、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 26/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=53ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mMで洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、8ml/minの流速で吸着したタンパク質の溶出を行った。各溶出画分について2S,4R-モナティン生成活性を検出したところ、約200mM NaCl相当の画分に2S,4R-モナティン生成活性が認められた。
2S,4R-モナティン生成活性が検出された画分を集めて、硫酸アンモニウム 1.5M、Tris-HCl 20mM(pH7.6)となるよう、硫酸アンモニウム、Tris-HCl(pH7.6)を添加した。この溶液を硫酸アンモニウム 1.5M、Tris-HCl 20mM(pH7.6)で平衡化した疎水性クロマトグラフィーカラムHiLoad 16/10 Phenyl Sepharose HP(GEヘルスケアバイオサイエンス社製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかった非吸着タンパク質を硫酸アンモニウム1.5M、Tris-HCl(pH7.6) 20mMを用いて洗い流した後、硫酸アンモニウム濃度を1.5Mから0Mまで直線的に変化させて、3ml/minの流速で2S,4R-モナティン生成酵素を溶出させた。得られた各溶出画分について2S,4R-モナティン生成活性を測定したところ、約0.8M硫酸アンモニウム相当の画分に2S,4R-モナティン生成活性が認められた。
2S,4R-モナティン生成活性が検出された画分を集めて、アミコン ウルトラ-15 10k(ミリポア)を用いて濃縮した。得られた濃縮液を、Tris-HCl(pH7.6) 20mM、NaCl 150mMで希釈した。Tris-HCl(pH7.6) 20mM、NaCl 150mMで平衡化されたゲルろ過カラムHiLoad 16/60 Superdex 200 pg(GEヘルスケアバイオサイエンス製、CV=120ml)に供し、1ml/minの流速で溶出した。この操作により分子量約100kDaと見積もられる位置で2S,4R-モナティン生成活性が確認された。
2S,4R-モナティン生成活性が検出された画分を集めて、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムMono Q 5/5(ファルマシア(GEヘルスケアバイオサイエンス)製、CV=1ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mMで洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、0.5ml/minの流速で吸着したタンパク質の溶出を行った。各溶出画分について2S,4R-モナティン生成活性を検出したところ、約300mM NaCl相当の画分に2S,4R-モナティン生成活性が認められた。
得られた画分をSDS-PAGEに供したところ、活性画分には47kDa付近にバンドが認められた。このバンドを、2S,4R-モナティンを生成するアミノトランスフェラーゼの候補としてN末端アミノ酸配列解析に供した。
実施例31で得られた精製酵素溶液をN末端アミノ酸配列解析に供したところ、AFLADILSRVKPSATIAVTQ(配列番号51)のN末端アミノ酸配列が得られた。N末端アミノ酸配列は、Agrobacterium tumefaciens str.C58由来アスパラギン酸アミノトランスフェラーゼ(AAK87940)と高い相同性を示した。
実施例31と同様の方法でRhizobium radiobacter AJ3976を培養した。得られた培養液から遠心分離により集菌し、ゲノムDNAを抽出した。
得られたゲノムDNAを鋳型にして、アミノトランスフェラーゼ遺伝子を含むDNA断片をPCR増幅した。プライマーは、Agrobacterium tumefaciens str.C58のゲノムDNA配列を参考にして、アミノトランスフェラーゼ遺伝子の上流100bpと、下流100bpのDNA配列から設計した、プライマーAg-u100-f(5’-ctggtgcagataagccggcttttgacc-3’:配列番号45)およびプライマーAg-d100-r(5’-ccaccttcatcatgctgctgtttctcg-3’:配列番号46)を用いた。PCRは、KOD-plus-ver.2(東洋紡)を用いて以下の条件で行った。
1 cycle 94℃、2min
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
実施例32で得られたN末端アミノ酸配列とも一致したため、2S,4R-モナティン生成活性を有するアミノトランスフェラーゼの遺伝子を取得できたと考えられた。
(1)Rhizobium sp.AJ12469由来アミノトランスフェラーゼ発現プラスミドの構築
Rhizobium sp.AJ12469のゲノムDNAを鋳型にして、Rhizobium sp.AJ12469由来アミノトランスフェラーゼ遺伝子を含むDNA断片をPCR増幅した。プライマーは、プライマー12469AT-Nde-f(5’-ggaattccatATGGCCTTCCTTGCCGACATTCTCT-3’:配列番号54)およびプライマー12469-xho-r(5’-actccgctcgagGCGGCAATCGGCGCAGAAACGCTGA-3’:配列番号55)を用いた。PCRは、KOD-plus-ver.2(東洋紡)を用いて以下の条件で行った。
1 cycle 94℃、2min
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
構築した発現プラスミドpET-22-12469AT-HisをE.coli BL21(DE3)に導入し、形質転換体をアンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen) 160mlに白金耳接種し、500ml容坂口フラスコを用いて37℃で16時間振盪させた。培養終了後、得られた培養液約1000mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMにて洗浄、懸濁し、4℃で30分間超音波破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
得られた可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMで平衡化したHis-tagタンパク質精製カラムHisPrep FF 16/10(ファルマシア(GEヘルスケアバイオサイエンス)製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 100mM、Imidozole 20mMで用いて洗い流した後、Imidazole濃度を20mMから250mMまで直線的に変化させて、3ml/minの流速で吸着したタンパク質の溶出を行った。
得られた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液を、Tris-HCl(pH7.6)20mMで希釈し、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 16/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6)20mMで用いて洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、3ml/minの流速で吸着したタンパク質の溶出を行った。
各溶出画分について2S,4R-モナティン生成活性を確認し、2S,4R-モナティン生成活性が認められた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液をTris-HCl(pH7.6) 20mMで希釈し、12469AT-His溶液とした。
(1)比色法でのL-Asp/α-KG、L-Asp/PA、およびL-Asp/(±)-MHOG活性測定
AJ12469LATの各種基質に対する活性測定を実施した。アミノ基転移反応のアミノドナー基質としては100mM L-Aspを用い、10mMの各種ケト酸に対する比活性を比色法で測定した。
L-Asp/α-KG活性:L-Asp-Na-1aq 100mM、α-KG-2Na 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH 2U/ml、25℃。340nmの減少から活性を算出。MDHはMalic Dehydrogenase from porcine heart(Sigma)を用いた。なお、L-Asp/α-KG活性は、表15において、アミノトランスフェラーゼ活性の項目「α-KG」に示す。
L-Asp/PA活性:L-Asp-Na-1aq 100mM、PA-Na 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH(上記と同様)2U/ml、25℃。340nmの減少から活性を算出。なお、L-Asp/PA活性は、表15において、アミノトランスフェラーゼ活性の項目「PA」に示す。
L-Asp/(±)-MHOG活性:L-Asp-Na-1aq 100mM、(±)-MHOG 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH(上記と同様) 2U/ml、LDH 10U/ml、25℃。340nmの減少から活性を算出。LDHはD-Lactate dehydrogenase from Leuconostoc mesenteroides(オリエンタル酵母)を用いた。(±)-MHOGに微量に混在しているPAを除くため、LDHを添加した。なお、L-Asp/(±)-MHOG活性は、表15において、アミノトランスフェラーゼ活性の項目「±MHOG」に示す。
目的活性である4R-IHOGからの2S,4R-モナティン生成活性、(±)-IHOGからの2S,4R-モナティン及び2S,4S-モナティン生成活性、及びIPAからのL-Trp副生活性をそれぞれ測定した。アミノ基転移反応のアミノドナー基質としては100mM L-Aspを用い、10mMの各種ケト酸に対するアミノ基転移反応を行い、生成したアミノ酸量をUPLCで定量し、比活性を算出した。
L-Asp/4R-IHOG活性:L-Asp-Na-1aq 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃。UPLC分析を行い、生成した2S,4R-モナティン、2S,4S-モナティンを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/4R-IHOG活性は、表15において、アミノトランスフェラーゼ活性の項目「4R-IHOG」に示す。
L-Asp/(±)-IHOG活性:L-Asp-Na-1aq 100mM、(±)-IHOG 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃。UPLC分析を行い、生成した2S,4R-モナティン、2S,4S-モナティンを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/(±)-IHOG活性は、表13において、アミノトランスフェラーゼ活性の項目「±IHOG」に示す。
L-Asp/IPA活性:L-Asp-Na-1aq 100mM、IPA 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM(pHは反応液作成後に1N NaOHで8.0に調整)、25℃。UPLC分析を行い、生成したTrpを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/IPA活性は、表15において、アミノトランスフェラーゼ活性の項目「IPA」に示す。
生成したモナティン及びTrpの定量はWaters社製 ACQUITY UPLCシステムを用いて定量した。測定条件を以下に示す。0.2mlで15分間反応を行ってから反応停止し、反応停止後の反応液を遠心し上清0.2ml程度をUPLC分析に供した。
12469-AT-Hisを用いて100mM L-Aspをアミノドナーとした際の10mM ケト酸に対する比活性測定結果を表15に示す。
実施例34で作製したpET-22-12469AT-His/E.coli BL21(DE3)をアンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen)3mlに白金耳接種し、試験管を用いて37℃で16時間振盪させた。培養終了後、得られた培養液1mlより菌体を遠心分離により集め、1mlのBugBuster Master Mix(Novagen)に懸濁した。得られた懸濁液を室温にて15分間静置して溶菌させ、遠心分離により菌体残渣を除き、得られた上清を可溶性画分とした。
得られた可溶性画分を用いて4R-IHOGからの2S,4R-モナティン合成反応を行った。反応液〔L-Asp-Na-1aq 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl 100mM、pH8.0〕 0.1mlに、上記の可溶性画分が0.05ml含まれるように加え、25℃で1時間反応させた。反応終了後、生成した2S,4R-モナティンを定量したところ、0.87mMであった。2S,4R-モナティンの定量は、UPLC分析により行った。分析条件は実施例29と同様である。
Corynebacterium ammoniagenes AJ1444の可溶性画分から、2S,4R-モナティンを生成するアミノトランスフェラーゼの精製を以下の通り行った。実施例25と同様にして、2S,4R-モナティン合成反応および2S,4R-モナティンの定量を行った。
LB寒天培地にCorynebacterium ammoniagenes AJ1444を塗布し、30℃で2日間培養した。
得られた菌体を酵素生産培地(酵母エキス 10g/l、トリプトン 10g/l、グルコース 1g/l、リン酸水素二カリウム 3g/l、リン酸二水素カリウム 1g/l、硫酸マグネシウム七水和物 0.1g/l、硫酸アンモニウム 5g/l)160mlに一白金耳接種し、500ml容坂口フラスコで30℃にて16時間振とう培養した。得られた培養液約1760mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mMにて洗浄、懸濁してガラスビーズを加え、マルチビーズショッカー(安井器械)にて菌体を破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
上記の可溶性画分に硫酸アンモニウム90%(w/w)を含むよう、硫酸アンモニウムを添加し、遠心分離により硫安沈殿を得た。
上記の硫安沈殿を、硫酸アンモニウム 1.0M、Tris-HCl 20mM(pH7.6)に懸濁した。この溶液を硫酸アンモニウム 1.0M、Tris-HCl 20mM(pH7.6)で平衡化した疎水性クロマトグラフィーカラムHiLoad 26/10 Phenyl Sepharose HP(GEヘルスケアバイオサイエンス社製、CV=53ml)に供して担体に吸着させた。担体に吸着しなかった非吸着タンパク質を硫酸アンモニウム 1.0M、Tris-HCl(pH7.6) 20mMを用いて洗い流した後、硫酸アンモニウム濃度を1.0Mから0Mまで直線的に変化させて、3ml/minの流速で2S,4R-モナティン生成酵素を溶出させた。得られた各溶出画分について2S,4R-モナティン生成活性を測定したところ、約0.2M硫酸アンモニウム相当の画分に2S,4R-モナティン生成活性が認められた。
2S,4R-モナティン生成活性が検出された画分を集めて、Tris-HCl(pH7.6) 20mMで一晩透析した。得られた溶液を、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 16/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mMで洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、2.25ml/minの流速で吸着したタンパク質の溶出を行った。各溶出画分について2S,4R-モナティン生成活性を検出したところ、約400mM NaCl相当の画分に2S,4R-モナティン生成活性が認められた。
2S,4R-モナティン生成活性が検出された画分を集めて、アミコン ウルトラ-15 10k(ミリポア)を用いて濃縮した。得られた濃縮液を、Tris-HCl(pH7.6) 20mM、NaCl 150mMで希釈した。Tris-HCl(pH7.6) 20mM、NaCl 150mMで平衡化されたゲルろ過カラムHiLoad 16/60 Superdex 200pg(GEヘルスケアバイオサイエンス製、CV=120ml)に供し、1.2ml/minの流速で溶出した。この操作により分子量約85kDaと見積もられる位置で2S,4R-モナティン生成活性が確認された。
2S,4R-モナティン生成活性が検出された画分を集めて、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムMono Q 5/5(ファルマシア(GEヘルスケアバイオサイエンス)製、CV=1ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mMで洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、1ml/minの流速で吸着したタンパク質の溶出を行った。各溶出画分について2S,4R-モナティン生成活性を検出したところ、約400mM NaCl相当の画分に2S,4R-モナティン生成活性が認められた。
得られた画分をSDS-PAGEに供したところ、活性画分には43kDa付近にバンドが認められた。このバンドを、2S,4R-モナティンを生成するアミノトランスフェラーゼの候補としてN末端アミノ酸配列解析に供した。
実施例37で得られた精製酵素溶液をN末端アミノ酸配列解析に供したところ、MSXIAQXILDQ(配列番号112)のN末端アミノ酸配列が得られた。N末端アミノ酸配列は、Corynebacterium striatum ATCC6940由来アスパラギン酸アミノトランスフェラーゼ(ZP_03935516)やCorynebacterium ammoniagenes DSM20306由来アスパラギン酸アミノトランスフェラーゼ(ZP_06838515)と高い相同性を示した。
実施例37と同様の方法でCorynebacterium ammoniagenes AJ1444を培養した。得られた培養液から遠心分離により集菌し、ゲノムDNAを抽出した。
得られたゲノムDNAを鋳型にして、アミノトランスフェラーゼ遺伝子を含むDNA断片をPCR増幅した。プライマーは、Corynebacterium ammoniagenes DSM20306のゲノムDNA配列を参考にして、アミノトランスフェラーゼ遺伝子の下流50bpのDNA配列から設計した、プライマーCo-d50-r(5’-cttccttggaacaagtcgaggaagac-3’:配列番号56)、およびCorynebacterium striatum ATCC6940由来アスパラギン酸アミノトランスフェラーゼ(ZP_03935516)とCorynebacterium ammoniagenes DSM20306由来アスパラギン酸アミノトランスフェラーゼ(ZP_06838515)のゲノムDNA配列で相同性の高い部分配列を参考にして設計した、プライマーCo-800-f(5’-gctatcgcacaattccaccgcacctt-3’:配列番号57)を用いた。PCRは、KOD-plus-ver.2(東洋紡)を用いて以下の条件で行った。
1 cycle 94℃、2min
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
1 cycle 94℃、2min
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
実施例38で得られたN末端アミノ酸配列とも一致したため、2S,4R-モナティン生成活性を有するアミノトランスフェラーゼの遺伝子を取得できたと考えられた。
(1)Corynebacterium ammoniagenes AJ1444由来アミノトランスフェラーゼ発現プラスミドの構築
Corynebacterium ammoniagenes AJ1444のゲノムDNAを鋳型にして、Corynebacterium ammoniagenes AJ1444由来アミノトランスフェラーゼ遺伝子を含むDNA断片をPCR増幅した。プライマーは、プライマー1444AT-Nde-f(5’-ggaattccatATGAGCCACATCGCTCAACGCATCC-3’:配列番号62)およびプライマー1444-xho-r(5’-actccgctcgagGGACTTTTCGAAGTATTGGCGAATG-3’:配列番号63)を用いた。PCRは、KOD-plus-ver.2(東洋紡)を用いて以下の条件で行った。
1 cycle 94℃、2min
25 cycles 98℃、10sec
55℃、10sec
68℃、60sec
1 cycle 68℃、60sec
4℃
構築した発現プラスミドpET-22-1444AT-HisをE.coli BL21(DE3)に導入し、形質転換体をアンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen) 160mlに白金耳接種し、500ml容坂口フラスコを用いて37℃で16時間振盪させた。培養終了後、得られた培養液約1000mlより菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidozole 10mMにて洗浄、懸濁し、4℃で30分間超音波破砕した。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
得られた可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidozole 10mMで平衡化したHis-tagタンパク質精製カラムHis TALON superflow 5ml Cartridge(Clontech)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 300mM、Imidozole 10mMで用いて洗い流した後、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidozole 150mMを用いて5ml/minの流速で吸着したタンパク質の溶出を行った。
得られた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液を、Tris-HCl(pH7.6)20mMで希釈し、Tris-HCl(pH7.6) 20mMで平衡化した陰イオン交換クロマトグラフィーカラムHiLoad 16/10 Q Sepharose HP(GEヘルスケアバイオサイエンス製、CV=20ml)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6)20mMで用いて洗い流した後、NaCl濃度を0mMから500mMまで直線的に変化させて、3ml/minの流速で吸着したタンパク質の溶出を行った。
各溶出画分について2S,4R-モナティン生成活性を確認し、2S,4R-モナティン生成活性が認められた画分を集めて、アミコン ウルトラ-15 30k(ミリポア)を用いて濃縮した。濃縮液をTris-HCl(pH7.6) 20mMで希釈し、1444AT-His溶液とした。
(1)比色法でのL-Asp/α-KG、L-Asp/PA、L-Asp/(±)-MHOG、L-Glu/PA、およびL-Glu/±MHOG活性測定
AJ1444LATの各種基質に対する活性測定を実施した。アミノ基転移反応のアミノドナー基質としては100mM L-AspまたはL-Gluを用い、10mMの各種ケト酸に対する比活性を比色法で測定した。
L-Asp/α-KG活性:L-Asp-Na-1aq 100mM、α-KG-2Na 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH 2U/ml、25℃。340nmの減少から活性を算出。MDHはMalic Dehydrogenase from porcine heart(Sigma)を用いた。なお、L-Asp/α-KG活性は、表17において、アミノトランスフェラーゼ活性の項目「α-KG」に示す。
L-Asp/PA活性:L-Asp-Na-1aq 100mM、PA-Na 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH(上記と同様)2U/ml、25℃。340nmの減少から活性を算出。なお、L-Asp/PA活性は、表17において、アミノトランスフェラーゼ活性の項目「PA」に示す。
L-Asp/(±)-MHOG活性:L-Asp-Na-1aq 100mM、(±)-MHOG 10mM、PLP 50μM、Tris-HCl(pH8.0)100mM、NADH 0.25mM、MDH(上記と同様)2U/ml、LDH 10U/ml、25℃。340nmの減少から活性を算出。LDHはD-Lactate dehydrogenase from Leuconostoc mesenteroides(オリエンタル酵母)を用いた。(±)-MHOGに微量に混在しているPAを除くため、LDHを添加した。なお、L-Asp/(±)-MHOG活性は、表17において、アミノトランスフェラーゼ活性の項目「±MHOG」に示す。
L-Glu/PA活性:L-Glu-Na 100mM、PA 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM、NH4Cl 100mM、NADH 0.25mM、GDH 10U/ml、25℃。340nmの減少から活性を算出。GDHはL-Glutamic Dehydrogenase from bovine liver(Sigma)を用いた。なお、L-Glu/PA活性は、表17において、アミノトランスフェラーゼ活性の項目「PA」に示す。
L-Glu/(±)-MHOG活性:L-Glu-Na 100mM、(±)-MHOG 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM、NH4Cl 100mM、NADH 0.25mM、GDH 10U/ml、25℃。340nmの減少から活性を算出。なお、L-Glu/(±)-MHOG活性は、表17において、アミノトランスフェラーゼ活性の項目「±MHOG」に示す。
目的活性である4R-IHOGからの2S,4R-モナティン生成活性、(±)-IHOGからの2S,4R-モナティン及び2S,4S-モナティン生成活性、及びIPAからのL-Trp副生活性をそれぞれ測定した。アミノ基転移反応のアミノドナー基質としては100mM L-AspまたはL-Gluを用い、10mMの各種ケト酸に対するアミノ基転移反応を行い、生成したアミノ酸量をUPLCで定量し、比活性を算出した。
L-Asp/4R-IHOG活性:L-Asp-Na-1aq 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃。UPLC分析を行い、生成した2S,4R-モナティン、2S,4S-モナティンを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/4R-IHOG活性は、表17において、アミノトランスフェラーゼ活性の項目「4R-IHOG」に示す。
L-Asp/(±)-IHOG活性:L-Asp-Na-1aq 100mM、(±)-IHOG 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃。UPLC分析を行い、生成した2S,4R-モナティン、2S,4S-モナティンを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/(±)-IHOG活性は、表17において、アミノトランスフェラーゼ活性の項目「±IHOG」に示す。
L-Asp/IPA活性:L-Asp-Na-1aq 100mM、IPA 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM(pHは反応液作成後に1N NaOHで8.0に調整)、25℃。UPLC分析を行い、生成したTrpを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Asp/IPA活性は、表17において、アミノトランスフェラーゼ活性の項目「IPA」に示す。
L-Glu/4R-IHOG活性:L-Glu-Na 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl(pH8.0) 100mM、25℃。UPLC分析を行い、生成した2S,4R-モナティン、2S,4S-モナティンを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Glu/4R-IHOG活性は、表17において、アミノトランスフェラーゼ活性の項目「4R-IHOG」に示す。
L-Glu/IPA活性:L-Glu-Na 100mM、IPA 10mM、PLP 50μM、Tris-HCl(pH8.0) 100mM(pHは反応液作成後に1N NaOHで8.0に調整)、25℃。UPLC分析を行い、生成したTrpを定量した。反応停止液は200mM クエン酸Na溶液(pH4.5)を用いた。なお、L-Glu/IPA活性は、表17において、アミノトランスフェラーゼ活性の項目「IPA」に示す。
1444-AT-Hisを用いて100mM L-Aspをアミノドナーとした際の10mM ケト酸に対する比活性測定結果を表17に示す。
実施例40で作製したpET-22-1444AT-His/E.coli BL21(DE3)をアンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen)3mlに白金耳接種し、試験管を用いて37℃で16時間振盪させた。培養終了後、得られた培養液1mlより菌体を遠心分離により集め、1mlのBugBuster Master Mix(Novagen)に懸濁した。得られた懸濁液を室温にて15分間静置して溶菌させ、遠心分離により菌体残渣を除き、得られた上清を可溶性画分とした。
得られた可溶性画分を用いて4R-IHOGからの2S,4R-モナティン合成反応を行った。反応液〔L-Asp-Na-1aq 100mM、4R-IHOG 10mM(微量の4S-IHOG含む)、PLP 50μM、Tris-HCl 100mM、pH8.0〕 0.1mlに、上記の可溶性画分が0.05ml含まれるように加え、25℃で15分間反応させた。反応終了後、生成した2S,4R-モナティンを定量したところ、0.13mMであった。2S,4R-モナティンの定量は、UPLC分析により行った。分析条件は実施例29と同様である。
精製した3976AT-His、12469AT-His、および1444AT-Hisを用いて、以下の条件で12時間反応を行った。反応は試験管を用いて1mlで行った。適宜サンプリングを行い、サンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL 遠心式フィルター10kDa(ミリポア)を用いて限外ろ過、ろ液を分析した。分析にはHPLCおよびキャピラリー電気泳動を用いた。
Ps_aadブロス:実施例17に記載の方法に従って調製した。
SpAld精製酵素:実施例19に記載の方法に従って調整した。
AJ3976LAT、AJ12469LAT、AJ1444LAT:実施例28、34、40に記載の方法に従って調製した。
OAA DCase:Oxaloacetate Decarboxylase from Pseudomonas sp.(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
SOD:Superoxide Dismutase from bovine liver(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
精製した3976AT-Hisを用いて、以下の条件で12時間反応を行った。反応は250ml容ミニジャーを用いて80mlで行った。適宜サンプリングを行い、サンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL 遠心式フィルター 10kDa(ミリポア)を用いて限外ろ過、ろ液を分析した。分析にはHPLCおよびキャピラリー電気泳動を用いた。
Ps_aadブロス:実施例17に記載の方法に従って調製した。
SpAld精製酵素:実施例19に記載の方法に従って調整した。
AJ3976LAT:実施例28に記載の方法に従って調製した。
OAA DCase:Oxaloacetate Decarboxylase from Pseudomonas sp.(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
SOD:Superoxide Dismutase from bovine liver(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
(1)in silico選抜アミノトランスフェラーゼ発現プラスミドの構築
in silicoから選抜した各種アミノトランスフェラーゼの遺伝子配列の5’末端にNdeI認識配列を、3’末端にXhoI認識配列を付与したDNA配列を、GenScript社のOptimumGene Codon Optimization Analysisに供し、E. coliでの遺伝子発現効率が最適化された合成DNAを得た。アミノトランスフェラーゼの種類は以下の通りである。
Deinococcus Geothermalis DSM 11300由来Putative Aminotransferase(Dge,ABF45244)(配列番号64、65)、Corynebacterium glutamicum R由来Hypothetical protein(Cgl,BAF53276)(配列番号66、67)、Thermus thermophilus HB27由来Lysn,Alpha-Aminoadipate Aminotransferase(TtHB,AAS80391)(配列番号68、69)、Thermotoga Maritima由来Aminotransferase,Putative(Tma1,AAD36207)(配列番号70、71)、Pyrococcus Horikoshii Ot3由来Human Kynurenine Aminotransferase II Homologue(PhoH,1X0M)(配列番号72、73)、Phormidium Lapideum由来Aspartate Aminotransferase(Pla,BAB86290)(配列番号74、75)、Thermus Thermophilus由来Aspartate Aminotransferase(Tth,BAD69869)(配列番号76、77)、Pyrococcus Horikoshii Ot3由来Aromatic Aminotransferase(PhoA,1DJU)(配列番号78、79)、Methanococcus jannaschii由来Mj0684(Mja,AAB98679)(配列番号80、81)、Thermotoga Maritima由来Aspartate Aminotransferase(Tma2,AAD36764)(配列番号82、83)、Saccharomyces cerevisiae由来Aspartate Aminotransferase(Sce,CAY81265)(配列番号84、85)、Eubacterium rectale由来Aspartate Aminotransferase(Ere,ACR74350)(配列番号86、87)、Bacillus pumilus SAFR-032由来Aspartate Aminotransferase(Bpu,ABV62783)(配列番号88、89)、Bacillus cellulosilyticus DSM 2522由来Putative transcriptional regulator, GntR family(Bce,ADU30616)(配列番号90、91)、Bacillus species(strain YM-2)由来Aspartate Aminotransferase aspC(Bsp,AAA22250)(配列番号92、93)、Sinorhizobium meliloti 1021由来Aspartate Aminotransferase aatB(SmeB,CAC47870)(配列番号94、95)、Methanothermobacter thermautotrophicus str.Delta H由来Branched-chain amino-acid aminotransferase(Mth,AAB85907)(配列番号96、97)、Lactobacillus acidophilus由来Aspartate aminotransferase(Lba,AAV43507)(配列番号98、99)、Sinorhizobium meliloti 1021由来Aspartate aminotransferase aatA(SmeA,CAC46904)(配列番号100、101)、Pyrococcus horikoshi OT3由来Hypothetical serine aminotransferase(PhoS,BAA30413)(配列番号102、103)、Thermoanaerobacter tengcongensis MB4由来PLP-dependent aminotransferases(Tte,AAM24436)(配列番号104、105)、Clostridium cellulolyticum H10由来Putative transcriptional regulator, GntR family(Cce,ACL75101)(配列番号106、107)、Rhodococcus erythropolis PR4由来Aspartate aminotransferase AspT(Rer,BAH31070)(配列番号108、109)、Saccharophagus degradans 2-40由来Transcriptional regulator(Sde,ABD82545)(配列番号110、111)。
構築した各種発現プラスミドpET-22-AT-HisをE.coli BL21(DE3)に導入し、形質転換体をアンピシリン100mg/lを含むOvernight Express Instant TB Medium(Novagen) 100mlに白金耳接種し、500ml容坂口フラスコを用いて16時間振盪させた。振盪温度は、Lbaは25℃、Dge、Pla、Tth、Tma2、Sce、Ere、Bpu、Bce、Bsp、SmeA、PhoS、Rer、Sdeは30℃、Cgl、TtHB、PhoH、PhoA、SmeB、Tte、Cceは37℃、Tma1、Mja、Mthは42℃で行った。培養終了後、得られた培養液より菌体を遠心分離により集め、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMにて洗浄、懸濁し、超音波破砕を行った。遠心分離により破砕液から菌体残渣を除き、得られた上清を可溶性画分とした。
得られた可溶性画分を、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMで平衡化したHis-tagタンパク質精製カラムHis TALON superflow 5ml Cartridge(Clontech)に供して担体に吸着させた。担体に吸着しなかったタンパク質(非吸着タンパク質)をTris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 10mMを用いて洗い流した後、Tris-HCl(pH7.6) 20mM、NaCl 300mM、Imidazole 150mMを用いて、5ml/minの流速で吸着したタンパク質の溶出を行った。得られた画分を集めて、アミコン ウルトラ-15 10k(ミリポア)を用いて濃縮した。濃縮液をTris-HCl(pH7.6) 20mMで希釈し、各種LAT溶液とした。必要に応じて、培養液量、His TALONカラムの連結数を増やして精製を行った。
精製した各種AT-Hisを用いて、以下の条件で15時間反応を行った。反応は試験管を用いて1mlで行った。反応終了後にサンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL 遠心式フィルター10kDa(ミリポア)を用いて限外ろ過、ろ液を分析した。分析にはHPLCおよびキャピラリー電気泳動を用いた。
Ps_aadブロス:実施例17に記載の方法に従って調製した。
SpAld精製酵素:実施例19に記載の方法に従って調整した。
各種LAT:実施例45に記載の方法に従って調製した。
OAA DCase:Oxaloacetate Decarboxylase from Pseudomonas sp.(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
SOD:Superoxide Dismutase from bovine liver(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
精製した各種AT-Hisを用いて、以下の条件で15時間反応を行った。反応は試験管を用いて1mlで行った。反応終了後にサンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL 遠心式フィルター10kDa(ミリポア)を用いて限外ろ過、ろ液を分析した。分析にはHPLCおよびキャピラリー電気泳動を用いた。
Ps_aadブロス:実施例17に記載の方法に従って調製した。
SpAld精製酵素:実施例19に記載の方法に従って調整した。
各種LAT:実施例45に記載の方法に従って調製した。
OAA DCase:Oxaloacetate Decarboxylase from Pseudomonas sp.(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
SOD:Superoxide Dismutase from bovine liver(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
Tth、Bpu、SmeA、Sdeの精製したAT-Hisを用いて、以下の条件で18時間反応を行った。反応は試験管を用いて1mlで行った。反応終了後にサンプルをTEバッファーで希釈し、アミコンウルトラ-0.5mL 遠心式フィルター10kDa(ミリポア)を用いて限外ろ過、ろ液を分析した。分析にはHPLCおよびキャピラリー電気泳動を用いた。
Ps_aadブロス:実施例17に記載の方法に従って調製した。
SpAld精製酵素:実施例19に記載の方法に従って調整した。
各種LAT:実施例45に記載の方法に従って調製した。
OAA DCase:Oxaloacetate Decarboxylase from Pseudomonas sp.(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
SOD:Superoxide Dismutase from bovine liver(Sigma)を用いた。酵素量(U)は、メーカー記載の値を用いた。
本明細書中に記載される、受託番号で特定された微生物は、その受託番号を参照することにより、所定の受託機関から入手可能である。表22に記載される微生物は、ブダペスト条約上の国際寄託機関である独立行政法人産業技術総合研究所特許生物寄託センター(日本国茨城県つくば市東1丁目1番地1 中央第6)に下記の日付で寄託され、下記の受託番号が付されている。なお、表22に記載されるように、これらの微生物は、異なる名称が従来付されていたが、再同定の結果、現在は以下のとおり分類されている。
配列番号2:Bacillus altitudinis由来のアミノトランスフェラーゼのアミノ酸配列
配列番号3:Bacillus altitudinis由来のアミノトランスフェラーゼ遺伝子(塩基番号231~1538)、ならびにその上流および下流領域のヌクレオチド配列
配列番号4:Bacillus altitudinis由来のアミノトランスフェラーゼ断片のアミノ酸配列
配列番号5:Bacillus altitudinis由来のアミノトランスフェラーゼ断片のアミノ酸配列
配列番号6:Bacillus altitudinis由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのフォワードプライマー(Bp-u200-f)
配列番号7:Bacillus altitudinis由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのリバースプライマー(Bp-d200-r)
配列番号8:Bacillus altitudinis由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのフォワードプライマー(1616AT-Nde-f)
配列番号9:Bacillus altitudinis由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのリバースプライマー(1616-xho-r)
配列番号10:Bacillus altitudinis由来のアミノトランスフェラーゼ遺伝子上に見出される、NdeIにより認識されるDNA配列を変換するためのフォワードプライマー(1616-delNde-f)
配列番号11:Bacillus altitudinis由来のアミノトランスフェラーゼ遺伝子上に見出される、NdeIにより認識されるDNA配列を変換するためのリバースプライマー(1616-delNde-r)
配列番号12:SpAld遺伝子を含むDNAフラグメントを増幅するためのフォワードプライマー(SpAld-f-NdeI)
配列番号13:SpAld遺伝子を含むDNAフラグメントを増幅するためのリバースプライマー(SpAld-r-HindIII)
配列番号14:SpAld遺伝子内のレアコドン6Lを変換するためのフォワードプライマー(6L-f)
配列番号15:SpAld遺伝子内のレアコドン6Lを変換するためのリバースプライマー(6L-r)
配列番号16:SpAld遺伝子内のレアコドン13Lを変換するためのフォワードプライマー(13L-f)
配列番号17:SpAld遺伝子内のレアコドン13Lを変換するためのリバースプライマー(13L-r)
配列番号18:SpAld遺伝子内のレアコドン18Pを変換するためのフォワードプライマー(18P-f)
配列番号19:SpAld遺伝子内のレアコドン18Pを変換するためのリバースプライマー(18P-r)
配列番号20:SpAld遺伝子内のレアコドン38Pを変換するためのフォワードプライマー(38P-f)
配列番号21:SpAld遺伝子内のレアコドン38Pを変換するためのリバースプライマー(38P-r)
配列番号22:SpAld遺伝子内のレアコドン50Pを変換するためのフォワードプライマー(50P-f)
配列番号23:SpAld遺伝子内のレアコドン50Pを変換するためのリバースプライマー(50P-r)
配列番号24:SpAld遺伝子内のレアコドン77P、81Pおよび84Rを変換するためのフォワードプライマー(77P-81P-84R-f)
配列番号25:SpAld遺伝子内のレアコドン77P、81Pおよび84Rを変換するためのリバースプライマー(77P-81P-84R-r)
配列番号26:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体K39Rを調製するためのフォワードプライマー(K39R_FW)
配列番号27:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体K39Rを調製するためのリバースプライマー(K39R_RV)
配列番号28:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体S258Gを調製するためのフォワードプライマー(S258G_FW)
配列番号29:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体S258Gを調製するためのリバースプライマー(S258G_RV)
配列番号30:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体T288Gを調製するためのフォワードプライマー(T288G_FW)
配列番号31:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体T288Gを調製するためのリバースプライマー(T288G_RV)
配列番号32:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体I289Aを調製するためのフォワードプライマー(I289A_FW)
配列番号33:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体I289Aを調製するためのリバースプライマー(I289A_RV)
配列番号34:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体Q287E/T288Gを調製するためのフォワードプライマー(Q287E/T288G_FW)
配列番号35:Bacillus altitudinis AJ1616由来のアミノトランスフェラーゼ変異体Q287E/T288Gを調製するためのリバースプライマー(Q287E/T288G_RV)
配列番号36:aspC遺伝子を破壊するためのDNAフラグメントを調製するためのプライマー(aspC-L1)
配列番号37:aspC遺伝子を破壊するためのDNAフラグメントを調製するためのプライマー(aspC-R1)
配列番号38:aspC遺伝子領域内のattL-cat-attRの挿入を確認するためのプライマー(aspC-up)
配列番号39:aspC遺伝子領域内のattL-cat-attRの挿入を確認するためのプライマー(attL-1)
配列番号40:aspC遺伝子領域内のattL-cat-attRの挿入を確認するためのプライマー(aspC-down)
配列番号41:aspC遺伝子領域内のattL-cat-attRの挿入を確認するためのプライマー(attR-1)
配列番号42:Pseudomonas putida由来のオキザロ酢酸デカルボキシラーゼ遺伝子のヌクレオチド配列
配列番号43:Pseudomonas putida由来のオキザロ酢酸デカルボキシラーゼ遺伝子のアミノ酸配列
配列番号44:Rhizobium radiobacter由来のアミノトランスフェラーゼ断片のアミノ酸配列
配列番号45:Agrobacterium tumefaciens str.C58由来のゲノムDNA配列に基づいて設計されたフォワードプライマー(Ag-u100-f)
配列番号46:Agrobacterium tumefaciens str.C58由来のゲノムDNA配列に基づいて設計されたリバースプライマー(Ag-d100-r)
配列番号47:Rhizobium radiobacter由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号48:Rhizobium radiobacter由来のアミノトランスフェラーゼのアミノ酸配列
配列番号49:Rhizobium radiobacter由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのフォワードプライマー(3976AT-Nde-f)
配列番号50:Rhizobium radiobacter由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのリバースプライマー(3976-xho-r)
配列番号51:Rhizobium sp.由来のアミノトランスフェラーゼ断片のアミノ酸配列
配列番号52:Rhizobium sp.由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号53:Rhizobium sp.由来のアミノトランスフェラーゼのアミノ酸配列
配列番号54:Rhizobium sp.由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのフォワードプライマー(12469AT-Nde-f)
配列番号55:Rhizobium sp.由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのリバースプライマー(12469-xho-r)
配列番号56:Corynebacterium ammoniagenes DSM20306由来のゲノムDNA配列に基づいて設計されたフォワードプライマー(Co-d50-r)
配列番号57:Corynebacterium striatum ATCC6940由来のアスパラギン酸アミノトランスフェラーゼ(ZP_03935516)およびCorynebacterium ammoniagenes DSM20306由来のアスパラギン酸アミノトランスフェラーゼに対応するゲノムDNA配列の間の相同領域に基づいて設計されたリバースプライマー
配列番号58:Corynebacterium ammoniagenes由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのフォワードプライマー(Co-890-r)
配列番号59:Corynebacterium ammoniagenes由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのリバースプライマー(Co-1060-r)
配列番号60:Corynebacterium ammoniagenes由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号61:Corynebacterium ammoniagenes由来のアミノトランスフェラーゼのアミノ酸配列
配列番号62:Corynebacterium ammoniagenes由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのフォワードプライマー(1444AT-Nde-f)
配列番号63:Corynebacterium ammoniagenes由来のアミノトランスフェラーゼ遺伝子を含むDNAフラグメントを増幅するためのリバースプライマー(1444-xho-r)
配列番号64:Deinococcus geothermalis由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号65:Deinococcus geothermalis由来のアミノトランスフェラーゼのアミノ酸配列
配列番号66:Corynebacterium glutamicum由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号67:Corynebacterium glutamicum由来のアミノトランスフェラーゼのアミノ酸配列
配列番号68:Thermus thermophilus由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号69:Thermus thermophilus由来のアミノトランスフェラーゼのアミノ酸配列
配列番号70:Thermotoga maritima由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号71:Thermotoga maritima由来のアミノトランスフェラーゼのアミノ酸配列
配列番号72:Pyrococcus horikoshii由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号73:Pyrococcus horikoshii由来のアミノトランスフェラーゼのアミノ酸配列
配列番号74:Phormidium lapideum由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号75:Phormidium lapideum由来のアミノトランスフェラーゼのアミノ酸配列
配列番号76:Thermus thermophilus由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号77:Thermus thermophilus由来のアミノトランスフェラーゼのアミノ酸配列
配列番号78:Pyrococcus horikoshii由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号79:Pyrococcus horikoshii由来のアミノトランスフェラーゼのアミノ酸配列
配列番号80:Methanococcus jannaschii由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号81:Methanococcus jannaschii由来のアミノトランスフェラーゼのアミノ酸配列
配列番号82:Thermotoga maritima由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号83:Thermotoga maritima由来のアミノトランスフェラーゼのアミノ酸配列
配列番号84:Saccharomyces cerevisiae由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号85:Saccharomyces cerevisiae由来のアミノトランスフェラーゼのアミノ酸配列
配列番号86:Eubacterium rectale由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号87:Eubacterium rectale由来のアミノトランスフェラーゼのアミノ酸配列
配列番号88:Bacillus pumilus由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号89:Bacillus pumilus由来のアミノトランスフェラーゼのアミノ酸配列
配列番号90:Bacillus cellulosilyticus由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号91:Bacillus cellulosilyticus由来のアミノトランスフェラーゼのアミノ酸配列
配列番号92:Bacillus sp. 由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号93:Bacillus sp. 由来のアミノトランスフェラーゼのアミノ酸配列
配列番号94:Sinorhizobium meliloti由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号95:Sinorhizobium meliloti由来のアミノトランスフェラーゼのアミノ酸配列
配列番号96:Methanothermobacter thermautotrophicus由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号97:Methanothermobacter thermautotrophicus由来のアミノトランスフェラーゼのアミノ酸配列
配列番号98:Lactobacillus acidophilus由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号99:Lactobacillus acidophilus由来のアミノトランスフェラーゼのアミノ酸配列
配列番号100:Sinorhizobium meliloti由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号101:Sinorhizobium meliloti由来のアミノトランスフェラーゼのアミノ酸配列
配列番号102:Pyrococcus horikoshii由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号103:Pyrococcus horikoshii由来のアミノトランスフェラーゼのアミノ酸配列
配列番号104:Thermoanaerobacter tengcongensis由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号105:Thermoanaerobacter tengcongensis由来のアミノトランスフェラーゼのアミノ酸配列
配列番号106:Clostridium cellulolyticum由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号107:Clostridium cellulolyticum由来のアミノトランスフェラーゼのアミノ酸配列
配列番号108:Rhodococcus erythropolis由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号109:Rhodococcus erythropolis由来のアミノトランスフェラーゼのアミノ酸配列
配列番号110:Saccharophagus degradans由来のアミノトランスフェラーゼ遺伝子のヌクレオチド配列
配列番号111:Saccharophagus degradans由来のアミノトランスフェラーゼのアミノ酸配列
配列番号112:Corynebacterium ammoniagenes由来のアミノトランスフェラーゼ断片のアミノ酸配列
Claims (30)
- L-アミノ酸の存在下において、4R-IHOGをL-アミノ酸アミノトランスフェラーゼに接触させて、2S,4R-モナティンを生成することを含む、2S,4R-モナティンまたはその塩の製造方法。
- L-アミノ酸アミノトランスフェラーゼの作用によりL-アミノ酸から生成したケト酸を、デカルボキシラーゼに接触させて分解することをさらに含む、請求項1記載の製造方法。
- L-アミノ酸がL-アスパラギン酸である、請求項1記載の製造方法。
- L-アミノ酸アミノトランスフェラーゼの作用によりL-アスパラギン酸から生成したオキサロ酢酸を、オキサロ酢酸デカルボキシラーゼに接触させて、ピルビン酸を不可逆的に生成することをさらに含む、請求項3記載の製造方法。
- L-アミノ酸アミノトランスフェラーゼが、アルスロバクター属、バチルス属、カンジダ属、コリネバクテリウム属、ロデロミセス属、マイクロコッカス属、マイクロバクテリウム属、ノカルディア属、シュードモナス属、リゾビウム属、ステノトロホモナス属、ディエジア属、オクロバクトラム属、ブレブンディモナス属、バークホルデリア属、カーニモナス属、ヤロウィア属、クロストリジウム属、デイノコッカス属、ユーバクテリウム属、ラクトバチルス属、メタノサーモバクター属、ホルミジウム属、ピロコッカス属、ロドコッカス属、サッカロマイセス属、サッカロファガス属、シノリゾビウム属、サーモアナエロバクター属、サーモトガ属、またはサーマス属に属する微生物に由来する、請求項1記載の製造方法。
- L-アミノ酸アミノトランスフェラーゼが、アルスロバクター・エスピー、バチルス・アルティトゥディニス、バチルス・セルロシリティカス、バチルス・プミルス、バチルス・エスピー、カンジダ・ノルベゲンシス、カンジダ・インコンスピクア、コリネバクテリウム・アンモニアゲネス、コリネバクテリウム・グルタミカム、ロデロミセス・エロンギスポルス、マイクロコッカス・ルテウス、マイクロバクテリウム・エスピー、ノカルディア・グロベルラ、シュードモナス・クロロラフィス、シュードモナス・シトロノクロリス、シュードモナス・フラギ、シュードモナス・プチダ、シュードモナス・シンキサンタ、シュードモナス・タエトロレンス、シュードモナス・エスピー、リゾビウム・ラディオバクター、リゾビウム・エスピー、ステノトロホモナス・エスピー、ディエジア・マリス、オクロバクトラム・シュードグリグノネンス、ブレブンディモナス・ディミヌタ、バークホルデリア・エスピー、カーニモナス・エスピー、ヤロウィア・リポリティカ、クロストリジウム・セルロリティカム、デイノコッカス・ゲオサーマリス、ユーバクテリウム・レクタル、ラクトバチルス・アシドフィルス、メタノサーモバクター・サーマウトトロフィカス、ホルミジウム・ラピデウム、ピロコッカス・ホリコシイ、ロドコッカス・エリスロポリス、サッカロマイセス・セレビジアエ、サッカロファガス・デグラダンス、シノリゾビウム・メリロティ、サーモアナエロバクター・テングコンゲネシス、サーモトガ・マリチマ、またはサーマス・サーモフィラスに属する微生物に由来する、請求項5記載の製造方法。
- L-アミノ酸アミノトランスフェラーゼが、配列番号2、配列番号48、配列番号53、配列番号61、配列番号65、配列番号67、配列番号69、配列番号73、配列番号75、配列番号77、配列番号83、配列番号85、配列番号87、配列番号89、配列番号91、配列番号93、配列番号95、配列番号97、配列番号99、配列番号101、配列番号103、配列番号105、配列番号107、配列番号109、または配列番号111により表されるアミノ酸配列に対して90%以上の同一性を示すアミノ酸配列からなる、請求項1記載の製造方法。
- L-アミノ酸アミノトランスフェラーゼが、配列番号2により表されるアミノ酸配列において39位、109位、128位、150位、258位、287位、288位、289位、303位、358位、及び431位のアミノ酸残基から選ばれる1個以上のアミノ酸残基の変異を含む、請求項7記載の製造方法。
- 1個以上のアミノ酸残基の変異が、以下からなる群より選ばれる1個以上のアミノ酸残基の置換である、請求項8記載の製造方法:
1)39位のリジンのアルギニンへの置換;
2)258位のセリンのグリシンへの置換;
3)287位のグルタミンのグルタミン酸への置換;
4)288位のスレオニンのグリシンへの置換;
5)289位のイソロイシンのアラニンへの置換;
6)109位のアスパラギン酸のグリシンへの置換;
7)150位のヒスチジンのチロシンへの置換;
8)303位のフェニルアラニンのロイシンへの置換;
9)358位のアスパラギン酸のチロシンへの置換;
10)431位のセリンのスレオニンへの置換;及び
11)128位のグルタミン酸のグリシンへの置換。 - L-アミノ酸アミノトランスフェラーゼを発現する形質転換体を用いて、4R-IHOGをL-アミノ酸アミノトランスフェラーゼに接触させる、請求項1記載の製造方法。
- インドール-3-ピルビン酸およびピルビン酸を縮合して、4R-IHOGを生成することをさらに含む、請求項1記載の製造方法。
- インドール-3-ピルビン酸およびピルビン酸をアルドラーゼに接触させることにより、インドール-3-ピルビン酸およびピルビン酸を縮合する、請求項11記載の製造方法。
- 4R-IHOGの生成に用いられるピルビン酸の少なくとも一部が、オキサロ酢酸デカルボキシラーゼの作用によりオキサロ酢酸から生成したピルビン酸に由来する、請求項11記載の製造方法。
- トリプトファンを脱アミノ化して、インドール-3-ピルビン酸を生成することをさらに含む、請求項11記載の製造方法。
- トリプトファンを脱アミノ化酵素に接触させることにより、トリプトファンを脱アミノ化する、請求項14記載の製造方法。
- 2S,4R-モナティンまたはその塩の製造が1つの反応槽中で行われる、請求項11または14記載の製造方法。
- 以下(I)および(II)を含む、2R,4R-モナティンまたはその塩の製造方法:
(I)請求項1記載の方法により、2S,4R-モナティンを生成すること;および
(II)2S,4R-モナティンを異性化させて、2R,4R-モナティンを生成すること。 - 芳香族アルデヒドの存在下において、2S,4R-モナティンを異性化する、請求項17記載の製造方法。
- 塩がナトリウム塩またはカリウム塩である、請求項17記載の製造方法。
- 下記(A)~(D)からなる群より選ばれるタンパク質である、L-アミノ酸アミノトランスフェラーゼ:
(A)配列番号2、配列番号48、配列番号53、または配列番号61により表されるアミノ酸配列からなるタンパク質;
(B)配列番号2、配列番号48、配列番号53、または配列番号61により表されるアミノ酸配列を含むタンパク質;
(C)配列番号2、配列番号48、配列番号53、または配列番号61により表されるアミノ酸配列に対して90%以上の同一性を示すアミノ酸配列からなり、かつL-アミノ酸アミノトランスフェラーゼ活性を有する、タンパク質;ならびに
(D)配列番号2、配列番号48、配列番号53、または配列番号61により表されるアミノ酸配列において、アミノ酸残基の欠失、置換、付加および挿入からなる群より選ばれる、1または数個のアミノ酸残基の変異を含むアミノ酸配列からなり、かつL-アミノ酸アミノトランスフェラーゼ活性を有する、タンパク質。 - L-アミノ酸アミノトランスフェラーゼが、配列番号2により表されるアミノ酸配列において39位、109位、128位、150位、258位、287位、288位、289位、303位、358位、及び431位のアミノ酸残基から選ばれる1個以上のアミノ酸残基の変異を含む、請求項20記載のL-アミノ酸アミノトランスフェラーゼ。
- 1個以上のアミノ酸残基の変異が、以下からなる群より選ばれる1個以上のアミノ酸残基の置換である、請求項21記載のL-アミノ酸アミノトランスフェラーゼ:
1)39位のリジンのアルギニンへの置換;
2)258位のセリンのグリシンへの置換;
3)287位のグルタミンのグルタミン酸への置換;
4)288位のスレオニンのグリシンへの置換;
5)289位のイソロイシンのアラニンへの置換;
6)109位のアスパラギン酸のグリシンへの置換;
7)150位のヒスチジンのチロシンへの置換;
8)303位のフェニルアラニンのロイシンへの置換;
9)358位のアスパラギン酸のチロシンへの置換;
10)431位のセリンのスレオニンへの置換;及び
11)128位のグルタミン酸のグリシンへの置換。 - 下記(a)~(e)からなる群より選ばれる、ポリヌクレオチド:
(a)配列番号1、配列番号47、配列番号52、または配列番号60により表されるヌクレオチド配列からなるポリヌクレオチド;
(b)配列番号1、配列番号47、配列番号52、または配列番号60により表されるヌクレオチド配列を含むポリヌクレオチド;
(c)配列番号1、配列番号47、配列番号52、または配列番号60により表されるアミノ酸配列に対して90%以上の同一性を示すヌクレオチド配列からなり、かつL-アミノ酸アミノトランスフェラーゼ活性を有するタンパク質をコードする、ポリヌクレオチド;
(d)配列番号1、配列番号47、配列番号52、または配列番号60により表されるヌクレオチド配列に対して相補的なヌクレオチド配列からなるポリヌクレオチドとストリンジェント条件下でハイブリダイズし、かつL-アミノ酸アミノトランスフェラーゼ活性を有するタンパク質をコードする、ポリヌクレオチド;ならびに
(e)請求項20記載のL-アミノ酸アミノトランスフェラーゼをコードするポリヌクレオチド。 - 請求項23記載のポリヌクレオチドを含む発現ベクター。
- 請求項24記載の発現ベクターが導入された形質転換体。
- 請求項25記載の形質転換体を培地中で培養して、L-アミノ酸アミノトランスフェラーゼを得ることを含む、L-アミノ酸アミノトランスフェラーゼの製造方法。
- L-アミノ酸の存在下において、4R-IHOGを請求項20記載のL-アミノ酸アミノトランスフェラーゼに接触させて、2S,4R-モナティンを生成することを含む、2S,4R-モナティンまたはその塩の製造方法。
- 以下(I’)および(II’)を含む、2R,4R-モナティンまたはその塩の製造方法:
(I’)請求項27記載の方法により、2S,4R-モナティンを生成すること;および
(II’)2S,4R-モナティンを異性化させて、2R,4R-モナティンを生成すること。 - 芳香族アルデヒドの存在下において、2S,4R-モナティンを異性化する、請求項28記載の製造方法。
- 塩がナトリウム塩またはカリウム塩である、請求項28記載の製造方法。
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JP2011551348A JPWO2012050125A1 (ja) | 2010-10-14 | 2011-10-12 | モナティンの製造方法 |
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WO2013073679A1 (ja) * | 2011-11-17 | 2013-05-23 | 味の素株式会社 | (2r,4r)モナティン多価金属塩結晶の製造方法 |
JP2018519828A (ja) * | 2015-07-02 | 2018-07-26 | シージェイ チェイルジェダン コーポレーション | 新規トランスアミナーゼ、及びそれを利用したアミノ化合物の脱アミン方法 |
JP2019189596A (ja) * | 2018-04-20 | 2019-10-31 | 上海中翊日化有限公司Shanghai Zhongyi Daily Chemical Co.,LTD | サーマス・サーモフィラスと酵母菌との組合せによる発酵生成物の用途 |
WO2021085405A1 (ja) * | 2019-10-28 | 2021-05-06 | 味の素株式会社 | ベンズアルデヒドの製造方法 |
WO2022075402A1 (ja) * | 2020-10-09 | 2022-04-14 | 株式会社カネカ | 大腸菌遺伝子改変株 |
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EP2479272A4 (en) | 2013-11-20 |
RU2012110249A (ru) | 2013-10-20 |
EP2479272A1 (en) | 2012-07-25 |
KR20120068872A (ko) | 2012-06-27 |
CN102741406A (zh) | 2012-10-17 |
RU2505606C2 (ru) | 2014-01-27 |
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